Prenylcysteine lyase (PCLase) catalyzes the cleavage of prenylcysteine (a protein modification) to form an isoprenoid aldehyde and the freed cysteine residue on the protein target. The FAD is non-covalently bound to PCLase. Not many mechanistic studies have been done looking at the reactions of the flavin, but the proposed mechanism is shown below. A hydride transfer from the C1 of the prenyl moiety to FAD is proposed, resulting in the reduction of the flavin to FADH 2 .  COformED IS a  carbocation  that is stabilized by the neighboring sulfur atom. FADH 2  then reacts with molecular oxygen to restore the oxidized enzyme. [ 23 ]


UDP-N-acetylenolpyruvylglucosamine Reductase (MurB) is an enzyme that catalyzes the  NADPH -dependent reduction of enolpyruvyl-UDP-N-acetylglucosamine (substrate) to the corresponding D-lactyl compound UDP-N-acetylmuramic acid (product). MurB is a monomer and contains one FAD molecule. Before the substrate can be converted to product, NADPH must first reduce FAD. Once NADP +  dissociates, the substrate can bind and the reduced flavin can reduce the product. [ 23 ]


Glutathione reductase  (GR) catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH). GR requires FAD and NADPH to facilitate this reaction; first a hydride must be transferred from NADPH to FAD. The reduced flavin can then act as a  nucleophile  to attack the disulfide, this forms the C4a-cysteine adduct. Elimination of this adduct results in a flavin-thiolate charge-transfer complex. [ 23 ]


Cytochrome P450  type enzymes that catalyze monooxygenase (hydroxylation) reactions are dependent on the transfer of two electrons from FAD to the P450. Two types of P450 systems are found in eukaryotes. The P450 systems that are located in the endoplasmic reticulum are dependent on a  cytochrome P-450 reductase  (CPR) that contains both an FAD and an  FMN . The two electrons on reduced FAD (FADH 2 ) are transferred one at a time to FMN and then a single electron is passed from FMN to the heme of the P450. [ 24 ]


The P450 systems that are located in the mitochondria are dependent on two electron transfer proteins: An FAD containing  adrenodoxin reductase  (AR) and a small iron-sulfur group containing protein named  adrenodoxin . FAD is embedded in the FAD-binding domain of AR. [ 25 ] [ 26 ]  The FAD of AR is reduced to FADH 2  by transfer of two electrons from NADPH that binds in the NADP-binding domain of AR. The structure of this enzyme is highly conserved to maintain precisely the alignment of electron donor NADPH and acceptor FAD for efficient electron transfer. [ 26 ]  The two electrons in reduced FAD are transferred one a time to adrenodoxin which in turn donates the single electron to the heme group of the mitochondrial P450. [ 27 ]


The structures of the reductase of the microsomal versus reductase of the mitochondrial P450 systems are completely different and show no homology. [ 24 ]


p -Hydroxybenzoate hydroxylase (PHBH) catalyzes the oxygenation of  p -hydroxybenzoate ( p OHB) to 3,4-dihyroxybenzoate (3,4-diOHB); FAD, NADPH and molecular oxygen are all required for this reaction. NADPH first transfers a hydride equivalent to FAD, creating FADH − , and then NADP +  dissociates from the enzyme. Reduced PHBH then reacts with molecular oxygen to form the flavin-C(4a)-hydroperoxide. The flavin hydroperoxide quickly hydroxylates  p OHB, and then eliminates water to regenerate oxidized flavin. [ 23 ]  An alternative flavin-mediated oxygenation mechanism involves the use of a  flavin-N(5)-oxide  rather than a flavin-C(4a)-(hydro)peroxide. [ 2 ] [ 3 ]


Chorismate synthase  (CS) catalyzes the last step in the  shikimate pathway —the formation of chorismate. Two classes of CS are known, both of which require  FMN , but are divided on their need for  NADPH  as a reducing agent. The proposed mechanism for CS involves radical species. The radical flavin species has not been detected spectroscopically without using a substrate analogue, which suggests that it is short-lived. However, when using a fluorinated substrate, a neutral flavin semiquinone was detected. [ 23 ]


Glutamate synthase catalyzes the conversion of 2-oxoglutarate into L-glutamate with L-glutamine serving as the nitrogen source for the reaction. All glutamate syntheses are iron-sulfur flavoproteins containing an iron-sulfur cluster and FMN. The three classes of glutamate syntheses are categorized based on their sequences and biochemical properties. Even though there are three classes of this enzyme, it is believed that they all operate through the same mechanism, only differing by what first reduces the FMN. The enzyme produces two glutamate molecules: one by the hydrolysis of glutamine (forming glutamate and ammonia), and the second by the ammonia produced from the first reaction attacking 2-oxoglutarate, which is reduced by FMN to glutamate. [ 23 ]


Due to the importance of  flavoproteins , it is unsurprising that approximately 60% of human flavoproteins cause human disease when mutated. [ 17 ]   In some cases, this is due to a decreased  affinity  for FAD or FMN and so excess riboflavin intake may lessen disease symptoms, such as for  multiple acyl-CoA dehydrogenase deficiency . [ 9 ]   In addition, riboflavin deficiency itself (and the resulting lack of FAD and FMN) can cause health issues. [ 9 ]  For example, in  ALS  patients, there are decreased levels of FAD synthesis. [ 9 ]   Both of these paths can result in a variety of symptoms, including developmental or gastrointestinal abnormalities, faulty  fat break-down ,  anemia , neurological problems,  cancer  or  heart disease ,  migraine , worsened vision and skin lesions. [ 9 ]   The pharmaceutical industry therefore produces riboflavin to supplement diet in certain cases.  In 2008, the global need for riboflavin was 6,000 tons per year, with production capacity of 10,000 tons. [ 4 ]  This $150 to 500 million market is not only for medical applications, but is also used as a supplement to animal food in the agricultural industry and as a  food colorant . [ 4 ]


New  design  of anti-bacterial medications is of continuing importance in scientific research as bacterial antibiotic resistance to common antibiotics increases.  A specific metabolic protein that uses FAD ( Complex II ) is vital for bacterial virulence, and so targeting FAD synthesis or creating FAD analogs could be a useful area of investigation. [ 28 ]  Already, scientists have determined the two structures FAD usually assumes once bound: either an extended or a butterfly conformation, in which the molecule essentially folds in half, resulting in the stacking of the adenine and isoalloxazine rings. [ 14 ]   FAD imitators that are able to bind in a similar manner but do not permit protein function could be useful mechanisms of inhibiting bacterial infection. [ 14 ]   Alternatively, drugs blocking FAD synthesis could achieve the same goal; this is especially intriguing because human and bacterial FAD synthesis relies on very different enzymes, meaning that a drug made to target bacterial FAD synthase would be unlikely to interfere with the human FAD synthase enzymes. [ 29 ]


Optogenetics  allows control of biological events in a non-invasive manner. [ 30 ]   The field has advanced in recent years with a number of new tools, including those to trigger light sensitivity, such as the Blue-Light-Utilizing FAD domains (BLUF).  BLUFs encode a 100 to 140  amino acid  sequence that was derived from photoreceptors in plants and bacteria. [ 30 ]   Similar to other  photoreceptors , the light causes structural changes in the BLUF domain that results in disruption of downstream interactions. [ 30 ]  Current research investigates proteins with the appended BLUF domain and how different external factors can impact the proteins. [ 30 ]


There are a number of molecules in the body that have native  fluorescence  including tryptophan,  collagen , FAD,  NADH  and  porphyrins . [ 31 ]   Scientists have taken advantage of this by using them to monitor disease progression or treatment effectiveness or aid in diagnosis.  For instance, native fluorescence of a FAD and NADH is varied in normal tissue and  oral submucous fibrosis , which is an early sign of invasive  oral cancer . [ 31 ]   Doctors therefore have been employing fluorescence to assist in diagnosis and monitor treatment as opposed to the standard  biopsy . [ 31 ]


5-Formamidoimidazole-4-carboxamide ribotide  (or  FAICAR ) is an intermediate in the formation of  purines .
It is formed by the enzyme  AICAR transformylase  from  AICAR  and  10-formyltetrahydrofolate . [ 1 ]


GC skew  is when the  nucleotides   guanine  and  cytosine  are over- or under-abundant in a particular region of  DNA  or  RNA . GC skew is also a statistical method for measuring strand-specific guanine overrepresentation. [ 1 ]


In equilibrium conditions (without  mutational  or  selective pressure  and with nucleotides randomly distributed within the  genome ) there is an equal frequency of the four DNA bases ( adenine ,  guanine ,  thymine , and  cytosine ) on both  single strands  of a DNA molecule. [ 2 ]  However, in most  bacteria  (e.g.  E. coli ) and some  archaea  (e.g.  Sulfolobus solfataricus ), nucleotide compositions are asymmetric between the  leading strand  and the  lagging strand : the leading strand contains more guanine (G) and thymine (T), whereas the lagging strand contains more adenine (A) and cytosine (C). [ 2 ]  This phenomenon is referred to as  GC  and  AT skew  and the corresponding statistics were defined [ 2 ]  as:


GC skew = (G - C)/(G + C)


AT skew = (A − T)/(A + T)


Erwin Chargaff's  work in 1950 demonstrated that, in DNA, the bases guanine and cytosine were found in equal abundance, and the bases adenine and thymine were found in equal abundance. However, there was no equality between the amount of one  pair  versus the other. [ 3 ]  Chargaff's finding is referred to as  Chargaff's rule  or  parity rule 2 . [ 3 ]   Three years later, Watson and Crick used this fact during their derivation of the structure of DNA, their  double helix model .


A natural result of parity rule 1, at the state of equilibrium, in which there is no mutation and/or selection biases in any of the two DNA strands, is that when there is an equal substitution rate, the  complementary nucleotides  on each strand have equal amounts of a given base and its complement. [ 4 ]  In other words, in each DNA strand the frequency of the occurrence of T is equal to A and the frequency of the occurrence of G is equal to C because the substitution rate is presumably equal. This phenomenon is referred to as  parity rule 2 . Hence, the second parity rule only exists when there is no mutation or substitution.


Any deviation from parity rule 2 will result in asymmetric base composition that discriminates the leading strand–i.e., the DNA strand that is replicated in the forward direction–from the lagging strand. This asymmetry is referred to as GC or AT skew. [ 2 ]


In some bacterial genomes, there is an enrichment of guanine over cytosine and thymine over adenine on the leading strand and vice versa for the lagging strand. The nucleotide composition skew spectra ranges from −1, which corresponds to G = 0 or A = 0, to +1, which corresponds to T= 0 or C = 0. [ 2 ]  Therefore, positive GC skew represents richness of G over C and the negative GC skew represents richness of C over G. As a result, one expects to see a positive GC skew and negative AT skew in the leading strand, and a negative GC skew and a positive AT skew in the lagging strand. [ 5 ]  GC or AT skew changes sign at the boundaries of the two  replichores , which correspond to DNA replication origin or terminus. [ 2 ] [ 4 ] [ 5 ]  Originally, this asymmetric nucleotide composition was explained as a different mechanism used in  DNA replication  between the leading strand and lagging strand. DNA replication is semi-conservative and an asymmetric process itself. [ 6 ]  This asymmetry is due to the formation of the replication fork and its division into nascent leading and lagging strands. The leading strand is synthesized continuously and in juxtapose to the leading strand; the lagging strand is replicated through short fragments of polynucleotide ( Okazaki fragments ) in a 5' to 3' direction. [ 6 ]


There are three major approaches to calculate and graphically demonstrate GC skew and its properties.


The first approach is GC and AT asymmetry. [ 2 ]  Jean R. Lobry was the first to report, in 1996, [ 7 ]  the presence of compositional asymmetry in the genomes of three bacteria:  E. coli ,  Bacillus subtilis , and  Haemophilus influenzae . The original formulas at the time were not called skew, but rather deviation from [A] = [T] or [C] = [G]:


deviation from [A] = [T] as (A − T)/(A + T);


deviation from [C] = [G] as (C − G)/(C + G);


where A, T, G, and C represent the frequency of occurrence of the equivalent base in a particular sequence in a defined length. A window sliding strategy is used to calculate deviation from C through the genome. In these plots, a positive deviation from C corresponds to lagging strand and negative deviation from C corresponds to leading strand. [ 8 ]  Furthermore, the site where the deviation sign switches corresponds to the origin or terminal. The x-axis represents the chromosome locations plotted 5′ to 3′ and y-axis represents the deviation value. The major weakness of this method is its window-size dependent property. Therefore, choosing an adequate window size greatly affects the outcome of the plot. Other techniques should be combined with deviation in order to identify and locate the origin of the DNA replication with greater accuracy.


The second approach is referred to as cumulative GC skew (CGC skew). [ 9 ]  This method still uses the sliding window strategy but it takes advantage of the sum of the adjacent windows from an arbitrary start. In this scheme, the entire genome is usually plotted 5' to 3' using an arbitrary start and arbitrary strand. In the cumulative GC skew plot, the peaks corresponds to the switch points (terminus or origin).


In contrast to Lobry's earlier paper, recent implementations of GC skew flips the original definition, redefining it to be:


GC skew = (G − C)/(G + C).


With the flipped definition of GC skew, the maximum value of the cumulative skew corresponds to the terminal, and the minimum value corresponds to the origin of replication.


The final approach is the  Z curve . [ 10 ]  Unlike the previous methods, this method do not uses the sliding window strategy and is thought to perform better as to finding the origin of replication. [ 10 ]  In this method, each base's  cumulative frequency  with respect to the base at the beginning of the sequence is investigated. The Z curve uses a three-dimensional representation with the following parameters:


x

 n

 =
 (

 A

 n

 +

 G

 n

 )
 −
 (

 C

 n

 +

 T

 n

 )

 {\displaystyle x_{n}=(A_{n}+G_{n})-(C_{n}+T_{n})}


y

 n

 =
 (

 A

 n

 +

 C

 n

 )
 −
 (

 G

 n

 +

 T

 n

 )

 {\displaystyle y_{n}=(A_{n}+C_{n})-(G_{n}+T_{n})}


z

 n

 =
 (

 A

 n

 +

 T

 n

 )
 −
 (

 C

 n

 +

 G

 n

 )

 {\displaystyle z_{n}=(A_{n}+T_{n})-(C_{n}+G_{n})}


Where

 n
 =
 0
 ,
 1
 ,
 2
 ,
 .
 .
 .
 N

 {\displaystyle n=0,1,2,...N}

 ,

 x

 n

 {\displaystyle x_{n}}

 represents the excess of purine over pyrimidine,

 y

 n

 {\displaystyle y_{n}}

 denotes excess of keto over amino, and

 z

 n

 {\displaystyle z_{n}}

 shows the relationship between the weak and strong  hydrogen bonds .

 x

 {\displaystyle x}

 and

 y

 {\displaystyle y}

 components can alone detect the  replication origin  and asymmetric composition of the strands.
A combination of these methods should be used for prediction of replication origin and terminal, in order to compensate for their weakness.


There is lack of consensus in scientific community with regard to the mechanism underlying the bias in nucleotide composition within each DNA strand. There are two major schools of thought that explain the mechanism behind the strand specific nucleotide composition in bacteria. [ 4 ]


The first one describes a bias and an asymmetric  mutational pressure  on each DNA strand during replication and  transcription . [ 4 ] [ 11 ]  Due to the asymmetric nature of the replication process, an unequal mutational frequency and  DNA repair  efficiency during the replication process can introduce more  mutations  in one strand as compared to the other. [ 5 ]  Furthermore, the time used for replication between the two strands varies and may lead to asymmetric mutational pressure between leading and lagging strand. [ 12 ]  In addition to mutations during DNA replication, transcriptional mutations can create strand specific nucleotide composition skew. [ 5 ]   Deamination  of cytosine and ultimately mutation of cytosine to thymine in one DNA strand can increase the relative number of guanine and thymine to cytosine and adenine. [ 5 ]  In most bacteria, the majority of the genes are encoded in the leading strand. [ 4 ]  For instance, the leading strand in  Bacillus   subtilis  encodes 75% of the genes. [ 5 ]  In addition, an excess of deamination and conversion of cytosine to thymine in the coding strand compared to the non-coding strand has been reported. [ 4 ] [ 5 ] [ 13 ]  One possible explanation is that the non-transcribed strand ( coding strand ) is single-stranded during the transcription process; therefore, it is more vulnerable to deamination compared to the transcribed strand ( non-coding strand ). [ 5 ] [ 14 ]  Another explanation is that the deamination repair activity during transcription does not occur on the coding strand. [ 5 ]  Only the transcribed strand benefits from these deamination repair events.


The second school of thought describes the mechanism of GC and AT skew as resulting from a difference in  selective pressure  between the leading and lagging strands. [ 4 ] [ 5 ] [ 14 ]  Examination of the prokaryotic genome shows a preference in third codon position for G over C and T over A. [ 5 ]  This discrimination creates an asymmetric nucleotide composition, if the coding strand is unequally distributed between the leading and lagging strands, as in the case for bacteria. In addition, the highly transcribed genes, such as  ribosomal proteins , have been shown to be located mostly on the leading strand in bacteria. [ 5 ]  Therefore, a bias in the third-position codon choice of G over C can lead to GC skew.  Additionally, some signal sequences are rich in guanine and thymine, such as  chi sequences , and these sequences might have a higher frequency of occurrence in one strand compared to the other. [ 4 ] [ 5 ]


Both mutational and selective pressure can independently introduce asymmetry in DNA strands. However, the combination and cumulative effect of both mechanisms is the most plausible explanation for GC and AT skew. [ 4 ] [ 14 ]


The GC skew is proven to be useful as the indicator of the DNA leading strand, lagging strand, replication origin, and replication terminal. [ 2 ] [ 4 ] [ 5 ]  Most bacteria and archaea contain only one DNA replication origin. [ 2 ]  The GC skew is positive and negative in the leading strand and in the lagging strand respectively; therefore, it is expected to see a switch in GC skew sign just at the point of DNA replication origin and terminus. [ 4 ]  GC skew can also be used to study the strand biases and mechanism related to them by calculating the excess of one base over its complementary base in different milieus. [ 4 ] [ 5 ] [ 14 ]  Method such as GC skew, CGC skew, and Z curve are tools that can provide opportunity to better investigate the mechanism of DNA replication in different organisms.


Glycineamide ribonucleotide  (or  GAR ) is a biochemical intermediate in the formation of  purine   nucleotides  via  inosine -5-monophosphate, and hence is a building block for  DNA  and  RNA . [ 1 ] [ 2 ] [ 3 ]  The vitamins  thiamine [ 4 ]  and  cobalamin [ 5 ]  also contain fragments derived from GAR. [ 6 ]


GAR is the product of the enzyme  phosphoribosylamine—glycine ligase  acting on  phosphoribosylamine  (PRA) to  combine  it with  glycine  in a process driven by  ATP . The reaction,  EC   6.3.4.13  forms an  amide  bond: [ 7 ]


The biosynthesis pathway next adds a  formyl  group from  10-formyltetrahydrofolate  to GAR, catalysed by  phosphoribosylglycinamide formyltransferase  in reaction  EC   2.1.2.2  and producing  formylglycinamide ribotide  (FGAR): [ 7 ]


GTPgammaS  (GTPγS, guanosine 5'-O-[gamma-thio]triphosphate) is a non-hydrolyzable or slowly hydrolyzable  G-protein -activating analog of  guanosine triphosphate  (GTP). Many GTP binding proteins demonstrate activity when bound to GTP, and are inactivated via the hydrolysis of the  phosphoanhydride  bond that links the γ-phosphate to the remainder of the nucleotide, leaving a bound  guanosine diphosphate  (GDP) and releasing an inorganic  phosphate .  This usually occurs rapidly, and the GTP-binding protein can then only be activated by exchanging the GDP for a new GTP molecule. [ 1 ]   The substitution of  sulfur  for one of the oxygens of the γ-phosphate of GTP creates a nucleotide that either cannot be hydrolyzed or is only slowly hydrolyzed.  This prevents the GTP-binding proteins from being inactivated, and allows the cellular processes that they carry out when active to be more easily studied. [ 2 ]


The consequences of the constitutive activation of GTP-binding proteins include stimulation of  phosphoinositide   hydrolysis , [ 3 ]   cyclic AMP  accumulation or elimination, [ 4 ]  and activation of specific  proto-oncogenes . [ 5 ]  The  35 S labelled radioligand of the compound,  35 SGTPγS, is used in  autoradiography  and G-protein binding studies. [ 6 ]


Guanosine diphosphate mannose  or  GDP-mannose  is a  nucleotide sugar  that is a substrate for  glycosyltransferase  reactions in  metabolism . This compound is a substrate for enzymes called  mannosyltransferases .


Known as donor of activated mannose in all glycolytic reactions, GDP-mannose is essential in eukaryotes. [ 1 ]


GDP-mannose is produced from  GTP  and  mannose-6-phosphate  by the enzyme  mannose-1-phosphate guanylyltransferase  (GDP-mannose pyrophosphorylase, GDP-MP). [ 2 ]  This enzyme belongs to a family of nucleotidyl-transferases and is a pervasive enzyme found in bacteria, fungi, plants, and animals. [ 3 ]


(p)ppGpp ,  guanosine pentaphosphate  and  tetraphosphate , also known as the  "magic spot" nucleotides , [ 1 ]  are  alarmones  involved in the  stringent response  in  bacteria  that cause the  inhibition  of  RNA synthesis  when there is a shortage of  amino acids . This inhibition by (p)ppGpp decreases  translation  in the cell, conserving  amino acids  present. Furthermore, ppGpp and pppGpp cause the  up-regulation  of many other genes involved in stress response such as the genes for amino acid uptake (from surrounding  media ) and  biosynthesis . [ 2 ]  (p)ppGpp is also conserved in  plants , where it is known to play a role in regulating growth and developmental processes.  [ 3 ]


ppGpp and pppGpp were first identified by Michael Cashel in 1969. [ 4 ]  These nucleotides were found to accumulate rapidly in  Escherichia coli  cells starved for amino acids and inhibit synthesis of ribosomal and transfer RNAs. [ 5 ]  It is now known that (p)ppGpp is also produced in response to other stressors including carbon and phosphate starvation. Historically, literature surrounding (p)ppGpp have given conflicting findings and information on its role in bacterial stress responses. [ 6 ]


E.coli  are shown to be more sensitive to accumulations of guanosine tetraphosphate than guanosine pentaphosphate.  [ 7 ]  A complete absence of (p)ppGpp causes multiple amino acid requirements, poor survival of aged cultures, aberrant cell division, morphology, and immotility, as well as being locked in a growth mode during entry into starvation.


The synthesis and degradation of (p)ppGpp have been most extensively characterized in the  bacterial   model organism   Escherichia coli.


(p)ppGpp is created via pppGpp  synthase , also known as RelA, and is converted from pppGpp to  ppGpp  via pppGpp phosphohydrolase. RelA is associated with about every one in two hundred  ribosomes  and it becomes activated when an uncharged  transfer RNA  (tRNA) molecule enters the A site of the ribosome, due to the shortage of amino acid required by the tRNA. If a  mutant  bacterium is  relA −  it is said to be relaxed and no regulation of RNA production due to amino acid absence is seen.


E. coli  produces a second protein responsible for degradation of (p)ppGpp,  SpoT . When the amino acid balance in the cell is restored, (p)ppGpp is  hydrolyzed  by  SpoT  and returned to a more energetically favorable state. This protein also has the capacity to synthesize (p)ppGpp, and seems to be the primary synthase under certain conditions of stress. Most other bacteria encode a single protein that is responsible for both synthesis and degradation of (p)ppGpp, generally homologs of SpoT.


Targets of (p)ppGpp include  rRNA   operons , of which there are seven in  E.coli , all of which have 2  promoters . When (p)ppGpp associates with the promoter it affects the  RNA polymerase   enzyme 's ability to bind and initiate  transcription . It is thought that (p)ppGpp may affect the stability of the open complex formed by RNA polymerase on DNA and therefore affect promoter clearance. Its presence also leads to an increase in pausing during transcription  elongation  and it competes with  nucleoside triphosphate   substrates .


There is now a consensus that (p)ppGpp is a determinant of growth rate control rather than nucleoside triphosphate (NTP) substrate concentrations.


ppGpp inhibits IF2-mediated fMet-Phe initiation dipeptide formation, probably by interfering with 30S and 50S subunit interactions. E. coli accumulates more ppGpp than pppGpp during amino acid starvation, and ppGpp has about 8-fold greater efficiency than that of pppGpp. While B. subtilis accumulates more pppGpp than ppGpp.


In E. coli amino acid starvation inhibited DNA replication at the initiation stage at oriC, most probably owing to the lack of the DnaA replication initiation protein. In B. subtilis, the replication arrest due to (p)ppGpp accumulation is caused by the binding of an  Rtp protein  to specific sites about 100-200kb away from oriC in both directions. DNA primase (DnaG) was directly inhibited by (p)ppGpp. Unlike E. coli, B. subtilis accumulates more pppGpp than ppGpp; the more abundant nucleotide is a more-potent DnaG inhibitor. ppGpp can bind with Obg protein which belongs to the conserved, small GTPase protein family. Obg protein interacts with several regulators (RsbT, RsbW, RsbX) necessary for the stress activation of sigma B.


The (p)ppGpp levels of the host seem to act as a sensor for phage lambda development, primarily affecting transcription. Modest ppGpp levels inhibit pR and active pE, pI, and paQ promoters in vivo and have effects in vitro that seem to favor lysogeny. In contrast, absent or high concentrations of (p)ppGpp favor lysis. Modest ppGpp levels favor lysogeny by leading to low HflB (FtsH). When ppGpp is either absent or high, HflB protease levels are high; this leads to lower CII (a lysogeny-promoting phage protein)
and favors lysis.


One of the key elements of promoters inhibited by (p)ppGpp is the presence of a GC-rich discriminator, defined as a region between TATA-box (-10 box) and +1 nt (where +1 is the transcription start site). Promoters negatively regulated by ppGpp have a 16-bp linker, in contrast with the 17-bp consensus. Promoters activated by ppGpp seem to have an AT-rich discriminator and linger linkers (for example, the  his  promoter linker is 18 bp).


Genetic evidence suggesting that RNAP was the target of ppGpp came from the discovery that M+ mutants (also called stringent RNAP mutants) display in vitro and in vivo mimicry of physiology and transcription regulation conferred by (p)ppGpp, even in its absence. Cross-linking ppGpp to RNAP reinforced this notion. Structural details of an association between ppGpp and RNAP came from the analysis of cocrystals that positioned ppGpp in the secondary channel of RNAP near the catalytic center.


DksA is a 17-kDa protein, its structure is similar to GreA and GreB, which are well-characterized  transcriptional elongation  factors. GreA and GreB bind directly to RNAP rather than DNA and act by inserting their N-terminal coiled-coil finger domain through the RNAP secondary channel. Two conserved acidic residues at the tip of the finger domain are necessary to induce RNAP's intrinsic ability to cleave backtracked RNA. DksA also possesses two acidic residues at its finger tip, but it does not induce nucleolytic cleavage activity. Instead, these residues are proposed to stabilize ppGpp binding to RNAP by mutual coordination of an Mg2+ ion that is crucial for polymerization.


ppGpp directly inhibits transcription from ribosomal promoters.  One model is ppGpp and DksA together and independently decrease the stability of the open complexes formed on DNA by RNAP.  Another model is the trapping mechanism. In this model, RNAP is trapped by ppGpp in closed complexes and is unable to initiate transcription. Thus, ppGpp seems to act at many levels, and the mechanism of its action is a complex outcome of several factors, intrinsic promoter properties not being the least of them. The transcription activation by ppGpp can be direct or indirect. Direct activation occurs when RNAP interacts with effectors, such as ppGpp, DksA or both, to increase transcription from a given promoter. Indirect activation by these effectors of one promoter relies on inhibition of other (strong) promoters, leading to increased availability of RNAP that indirectly activates transcription initiation. The promoters that activated directly by ppGpp include P argI , P thrABC , P livJ , and P hisG . The indirectly activation promoters include these dependent on sigma factors: S, H, N, E. When strong promoters, such as  rrn , are inhibited, there more RNAP are available for these alternative sigma factors.


When (p)ppGpp is absent, pathogenicity is compromised for reasons that vary with the organism studied. Deleting  rel A and  spo T genes, but not  rel A alone, gave a (p)ppGpp 0  state that resulted in strong attenuation in mice and noninvasiveness in vitro. Vaccine tests reveal that 30 days after single immunization with the (p)ppGpp 0  strain, mice were protected from challenge with wild-type Salmonella at a dose 10 6 -fold above the established LD 50 .


It was proposed that increased synthesis of (p)ppGpp would cause  polyphosphate  (PolyP) accumulation in  E. coli . [ 8 ]  The alarmone could interact with  exopolyphosphatase PPX , which would inhibit the hydrolysis of PolyP, thus causing its accumulation in bacteria. Although it has recently been shown that it is actually DksA and not (p)ppGpp that causes this buildup. [ 9 ]   It has been shown in  Pseudomonas aeruginosa  that the  phoU  mutant ( phoU  belongs to the  Pho Regulon ) synthesizes more (p)ppGpp and this would be one of the reasons that it accumulates more polyphosphate. [ 10 ]


Guanosine monophosphate  ( GMP ), also known as  5′-guanidylic acid  or  guanylic acid  ( conjugate base   guanylate ), is a  nucleotide  that is used as a  monomer  in  RNA . It is an  ester  of  phosphoric acid  with the  nucleoside   guanosine . GMP consists of the  phosphate   group , the  pentose   sugar   ribose , and the  nucleobase   guanine ; hence it is a  ribonucleotide  monophosphate. Guanosine monophosphate is commercially produced by microbial fermentation. [ 1 ]


As an  acyl   substituent , it takes the form of the prefix  guanylyl- .


GMP synthesis starts with  D -ribose 5′-phosphate, a product of the pentose phosphate pathway. The synthesis proceeds by the gradual formation of the purine ring on carbon-1 of ribose, with CO 2 , glutamine, glycine, aspartate and one-carbon derivatives of tetrahydrofolate donating various elements towards the building of the ring. [ 2 ]


As inhibitor of guanosine monophosphate synthesis in experimental models, the  glutamine  analogue  DON  can be used. [ 3 ]


GMP can also exist as a cyclic structure known as  cyclic GMP . Within certain cells the enzyme guanylyl cyclase makes cGMP from GTP.


cGMP plays an important role in mediating hormonal signaling. [ 2 ]


GMP was originally identified as the umami substance in dried  shiitake  mushroom. The drying process significantly increases GMP content with the breakdown of RNA. It can be found in a number of other mushrooms. [ 4 ]


Industrial production is based on fermentation: a bacterium converts sugars into  AICA ribonucleotide , which is then converted chemically to GMP. [ 5 ]   Tapioca starch  is a possible sugar source. [ 6 ]


Guanosine monophosphate is known as  E number  reference E626. [ 7 ]  In the form of its  salts , such as  disodium guanylate  ( E627 ),  dipotassium guanylate  (E628) and  calcium guanylate  (E629), are  food additives  used as  flavor enhancers  to provide the  umami  taste. [ 7 ]  It is often used in synergy with  disodium inosinate ; the combination is known as  disodium 5′-ribonucleotides . Disodium guanylate is often found in instant noodles, potato chips and snacks, savoury rice, tinned vegetables, cured meats, and packet soup.


As it is a fairly expensive additive, it is usually not used independently of  glutamic acid  or  monosodium glutamate  (MSG), which also contribute umami.  If inosinate and guanylate salts are present in a list of ingredients but MSG does not appear to be, the glutamic acid is likely provided as part of another ingredient, such as a processed soy protein complex (hydrolyzed soy protein),  autolyzed yeast , or  soy sauce .


Guanosine diphosphate , abbreviated  GDP , is a  nucleoside diphosphate . It is an  ester  of  pyrophosphoric acid  with the  nucleoside   guanosine . GDP consists of a  pyrophosphate   group , a  pentose   sugar   ribose , and the  nucleobase   guanine . [ 1 ]


GDP is the product of  GTP   dephosphorylation  by  GTPases , e.g., the  G-proteins  that are involved in  signal transduction .


GDP is converted into GTP with the help of  pyruvate kinase  and phosphoenolpyruvate.


The hydrolysis of GTP to GDP is facilitated by  GTPase  enzymes, which utilize a conserved active site motif known as the  GTPase-activating protein  (GAP). Initially, a water molecule is coordinated by the active site residues of the GTPase enzyme. The water molecule attacks the γ-phosphate of GTP, leading to the formation of a pentavalent transition state. This transition state is stabilized by interactions with the active site residues, including conserved catalytic residues. As a result, the γ-phosphate is cleaved, and inorganic phosphate (Pi) is released. This step also causes a conformational change in the enzyme that promotes the release of GDP. [ 2 ]


GDP is involved in intracellular signaling processes functioning as a critical regulator in the activity of GTPases. GTPases act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. The interconversion between GDP and GTP is tightly controlled and serves as a molecular timer for signal transduction pathways. When an extracellular signal triggers the activation of a  G-protein coupled receptor  (GPCR), the associated G-protein exchanges its bound GDP for GTP, leading to a conformational change and activation of downstream signaling cascades. [ 3 ]  This activation can stimulate a variety of cellular responses, including modulation of gene expression, cytoskeletal rearrangements, and regulation of enzymatic activities. The hydrolysis of GTP to GDP by the GTPase activity of the G-protein restoring the inactive state, thus terminates the signaling event. [ 4 ]


In a type of intracellular signaling referred to as the Nuclear Import process, cytosolic proteins like alpha and beta importins would bind the nuclear localization sequence that is located within the protein, to which this complex would bind to a nuclear pore complex that allows for translocation into the nucleus. [ 5 ]  RanGTPase would bind to the beta importin which removes it from the complex, followed by the removal of the alpha importin which is also bound to the RanGTPase. While bound to the RanGTPase, the importins leave the nucleus where the Ran is hydrolyzed, converting the bound GTP into GDP. Ran-GDP can return the nucleus through its bind to a nuclear transport factor, where a Guanine Nucleotide Exchange Factor Protein can come to catalyze the exchange of GTP for GDP. We also see GTP hydrolysis in situations with Ras proteins in which the same reaction occurs. [ 6 ]


Guanosine-5'-triphosphate  ( GTP ) is a  purine   nucleoside triphosphate .  It is one of the building blocks needed for the synthesis of  RNA  during the  transcription  process. Its structure is similar to that of the  guanosine   nucleoside , the only difference being that  nucleotides  like GTP have  phosphates  on their  ribose  sugar. GTP has the  guanine   nucleobase  attached to the 1' carbon of the ribose and it has the triphosphate moiety attached to ribose's 5' carbon.


It also has the role of a source of energy or an activator of substrates in metabolic reactions, like that of  ATP , but more specific. It is used as a source of energy for  protein synthesis  and  gluconeogenesis .


GTP is essential to  signal transduction , in particular with  G-proteins , in second-messenger mechanisms where it is converted to  guanosine diphosphate  (GDP) through the action of  GTPases .


GTP is involved in energy transfer within the cell.  For instance, a GTP molecule is generated by one of the enzymes in the  citric acid cycle . This is tantamount  to the generation of one molecule of  ATP , since GTP is readily converted to ATP with  nucleoside-diphosphate kinase  (NDK). [ 1 ]


During the elongation stage of  translation , GTP is used as an energy source for the binding of a new amino-bound  tRNA  to the A site of the  ribosome . GTP is also used as an energy source for the translocation of the ribosome towards the 3' end of the  mRNA . [ 2 ]


During  microtubule  polymerization, each heterodimer formed by an alpha and a beta tubulin molecule carries two GTP molecules, and the GTP is hydrolyzed to GDP when the tubulin dimers are added to the plus end of the growing microtubule. Such GTP hydrolysis is not mandatory for microtubule formation, but it appears that only GDP-bound tubulin molecules are able to depolymerize. Thus, a GTP-bound tubulin serves as a cap at the tip of microtubule to protect from depolymerization; and, once the GTP is hydrolyzed, the microtubule begins to depolymerize and shrink rapidly. [ 3 ]


The translocation of proteins into the mitochondrial matrix involves the interactions of both GTP and ATP. The importing of these proteins plays an important role in several pathways regulated within the mitochondria organelle, [ 4 ]  such as converting  oxaloacetate  to  phosphoenolpyruvate  (PEP) in gluconeogenesis. [ citation needed ]


GTP, in combination with  ribulose 5-phosphate , are the precursor compounds for the synthesis of  riboflavin  (vitamin B 2 ). [ 5 ]


In the cell, GTP is synthesised through many processes including:


5'-Guanylyl imidodiphosphate  ( GDPNP ) is a  purine   nucleotide .  It is an analog of  guanosine triphosphate  in which one of the oxygen atoms is replaced with an  amine , producing a  non-hydrolyzable   functional group . Guanylyl imidodiphosphate binds tightly to  G-proteins  in the presence of Mg 2+ . [ 2 ]  Guanylyl imidodiphosphate is a potent stimulator of  adenylate cyclase . [ 2 ]   It is often used in studies of protein synthesis. [ 3 ] [ 4 ]


Hovlinc RNA  is a self-cleaving  ribozyme  of about 168  nucleotides  found in a very  long noncoding RNA  (vlincRNA) in humans, chimpanzees, and gorillas. [ 1 ]  The word "hovlinc" comes from "hominin vlincRNA-located" RNA. Hovlinc is only a fourth known case of a ribozyme in human. Self-cleavage activity of Hovlinc has been shown in human, chimpanzees and bonobos, but is absent in gorillas, raising questions about Hovlinc's biological function and  evolution .


There are only a few known examples of ribozymes in human, [ 2 ]  including Hovlinc,  Mammalian CPEB3 ribozyme ,  Hammerhead ribozyme  (HH9 and HH10 [ 3 ] ) and B2 SINE ribozyme. [ 4 ]  Presumably Hovlinc acquired its self-cleaving activity about 10 to 13 million of years ago, which coincides with the last common ancestor of humans, gorillas, and chimpanzees. Hovlinc presents catalytic activity in  hominids  but not in gorillas where a  mutation  abolished the self-cleavage activity.


Hovlinc is a very structured RNA that contains four  stem loops  joined in a central loop, it also features large  pseudoknots  that help to bring together the second and fourth helices, which helps the RNA to get the more compacted structure that allows catalytic activity.


Inosine triphosphate  ( ITP ) is an intermediate in the  purine metabolism  pathway, seen in the synthesis of  ATP  and  GTP . It comprises an inosine  nucleotide  containing three phosphate groups esterified to the sugar  moiety .


ITP results from  deamination  of  ATP . Incorporation of ITP into the DNA from the  nucleotide  pool can lead to DNA damage, mutagenesis and other harmful effects. [ 1 ]  ITP is processed by the enzyme  inosine triphosphate pyrophosphatase  (ITPA), which turns it into  inosine monophosphate  (IMP), to avoid incorporation into DNA. [ 1 ]


Inosinic acid  or  inosine monophosphate  ( IMP ) is a  nucleotide  (that is, a  nucleoside  monophosphate). Widely used as a flavor enhancer, it is typically obtained from chicken byproducts or other  meat industry  waste. Inosinic acid is important in  metabolism . It is the  ribonucleotide  of  hypoxanthine  and the first nucleotide formed during the synthesis of  purine  nucleotides. It can also be formed by the  deamination  of  adenosine monophosphate  by  AMP deaminase . It can be  hydrolysed  to  inosine .


The enzyme deoxyribonucleoside triphosphate pyrophosphohydrolase, encoded by YJR069C in  Saccharomyces cerevisiae  and containing (d)ITPase and (d)XTPase activities, hydrolyzes  inosine triphosphate  (ITP) releasing  pyrophosphate  and IMP. [ 1 ]


Important derivatives of inosinic acid include the purine nucleotides found in  nucleic acids  and  adenosine triphosphate , which is used to store  chemical energy  in  muscle  and other tissues.


In the food industry, inosinic acid and its  salts  such as  disodium inosinate  are used as  flavor enhancers . It is known as  E number  reference  E630 .


The inosinate synthesis is complex, beginning with a  5-phosphoribosyl-1-pyrophosphate  (PRPP).  Enzymes  taking part in IMP synthesis constitute a multienzyme complex in the cell. Evidence demonstrates that there are multifunctional enzymes, and some of them catalyze non-sequential steps in the pathway. [ citation needed ]


Within a few steps inosinate becomes  AMP  or  GMP . [ 2 ]  Both compounds are  RNA   nucleotides . [ 2 ]  AMP differs from inosinate by the replacement of IMP's carbon-6 carbonyl with an amino group. The interconversion of AMP and IMP occurs as part of the  purine nucleotide cycle . [ 3 ]  GMP is formed by the inosinate oxidation to  xanthylate  (XMP), and afterwards adds an amino group on carbon 2. Hydrogen acceptor on inosinate oxidation is  NAD+ . Finally, carbon 2 gains the amino group by spending an ATP molecule (which becomes AMP+2Pi). While AMP synthesis requires GTP, GMP synthesis uses ATP. That difference offers an important regulation possibility.


Inosinate and many other molecules inhibit the synthesis of 5- phosphoribosylamine  from 5-phosphoribosyl-1-pyrophosphate (PRPP), disabling the enzyme that catalyzes the reaction: glutamine-5-phosphoribosyl-1-pyrophosphate-amidotransferase. In other words, when levels of inosinate are high, glutamine-5-phosphoribosyl-1-pyrophosphate-amidotransferase is inhibited, and, as a consequence, inosinate levels decrease. Also, as a result, adenylate and guanylate are not produced, which means that RNA synthesis cannot be completed because of the lack of these two important RNA nucleotides.


Inosinic acid can be converted into various  salts  including  disodium inosinate  (E631),  dipotassium inosinate  (E632), and  calcium inosinate  (E633). These three compounds are used as  flavor enhancers  for the basic taste  umami  or savoriness with a comparatively high effectiveness. They are mostly used in soups, sauces, and seasonings for the intensification and balance of the flavor of meat.


An  internal ribosome entry site , abbreviated  IRES , is an  RNA  element that allows for  translation  initiation in a cap-independent manner, as part of the greater process of  protein synthesis . Initiation of  eukaryotic translation  nearly always occurs at and is dependent on the  5' cap  of mRNA molecules, where the translation initiation complex forms and  ribosomes  engage the mRNA. IRES elements, however allow ribosomes to engage the mRNA and begin translation independently of the 5' cap.


IRES sequences were first discovered in 1988 in the  poliovirus  (PV) and encephalomyocarditis virus (EMCV) RNA genomes in the laboratories of  Nahum Sonenberg [ 1 ]  and  Eckard Wimmer , [ 2 ]  respectively. They are described as distinct regions of RNA molecules that are able to recruit the  eukaryotic   ribosome  to the mRNA. This process is also known as cap-independent translation. It has been shown that IRES elements have a distinct  secondary  or even  tertiary structure , but similar structural features at the levels of either  primary  or secondary structure that are common to all IRES segments have not been reported to date.


Use of IRES sequences in molecular biology soon became common as a tool for expressing multiple genes from a single transcriptional unit in a genetic  vector . In such vectors, translation of the first cistron is initiated at the 5' cap, and translation of any downstream cistron is enabled by an IRES element appended at its 5' end. [ 3 ]


IRES elements are most commonly found in the  5' untranslated region , but may also occur elsewhere in mRNAs. The mRNA of viruses of the  Dicistroviridae  family possess two open reading frames (ORFs), and translation of each is directed by a distinct IRES. It has also been suggested that some  mammalian  cellular mRNAs also have IRESs, although this has been a matter of dispute. [ 4 ] [ 5 ]  A number of these cellular IRES elements are located within mRNAs encoding proteins involved in  stress   survival , and other processes critical to survival. As of September 2009, there are 60 animal and 8 plant viruses reported to contain IRES elements and 115 mRNA sequences containing them as well. [ 6 ]


IRESs are often used by viruses as a means to ensure that viral translation is active when host translation is inhibited. These mechanisms of host translation inhibition are varied, and can be initiated by both virus and host, depending on the type of virus. However, in the case of most picornaviruses, such as  poliovirus , this is accomplished by viral proteolytic cleavage of  eIF4G  so that it cannot interact with the  5'cap  binding protein  eIF4E . Interaction between these two  eukaryotic initiation factors  (eIFs) of the  eIF4F  complex is necessary for  40S ribosomal subunit  recruitment to the 5' end of mRNAs, which is further thought to occur with mRNA  5'cap  to 3'  poly(A) tail  loop formation. The virus may even use partially-cleaved  eIF4G  to aid in initiation of IRES-mediated translation.


Cells may also use IRESs to increase translation of certain proteins during  mitosis  and  programmed cell death . In mitosis, the cell dephosphorylates  eIF4E  so that it has little affinity for the  5'cap . As a result, the  40S ribosomal subunit , and the translational machinery is diverted to IRES within the mRNA. Many proteins involved in mitosis are encoded by IRES mRNA. In programmed cell death, cleavage of eIF-4G, such as performed by viruses, decreases translation. Lack of essential proteins contributes to the death of the cell, as does translation of IRES mRNA sequences coding proteins involved in controlling cell death. [ 7 ]


To date, the mechanism of viral IRES function is better characterized than the mechanism of cellular IRES function, [ 8 ]  which is still a matter of debate.  HCV -like IRESs directly bind the  40S  ribosomal subunit to position their initiator codons are located in  ribosomal P-site  without mRNA scanning. These IRESs still use the  eukaryotic initiation factors  (eIFs)  eIF2 ,  eIF3 ,  eIF5 , and  eIF5B , but do not require the factors  eIF1 ,  eIF1A , and the  eIF4F  complex. In contrast,  picornavirus  IRESs do not bind the 40S subunit directly, but are recruited instead through the  eIF4G -binding site. [ 9 ]  Many viral IRES (and cellular IRES) require additional proteins to mediate their function, known as IRES  trans -acting factors (ITAFs). The role of ITAFs in IRES function is still under investigation.


Testing of sequences for potential IRES function has generally relied on the use of  bicistronic  reporter  assays . In these tests, a candidate IRES segment is introduced into a plasmid between two  cistrons  encoding two different reporter proteins.  A promoter upstream of the first cistron drives transcription of both cistrons in a single mRNA.  Cells are transfected with the plasmid and assays are subsequently performed to quantitate expression of the two reporters in the cells. An increase in the ratio of expression of the downstream reporter relative to the upstream reporter is taken as evidence for IRES activity in the test sequence.  However, without characterization of the mRNA species produced from such plasmids, other explanations for the increase in this ratio cannot be ruled out. [ 4 ] [ 5 ]  For example, there are multiple known cases of suspected IRES elements that were later reported as having  promoter  function.  Unexpected  splicing  activity within several reported IRES elements have also been shown to be responsible for the apparent IRES function observed in bicistronic reporter tests. [ 10 ]   A promoter or splice acceptor within a test sequence can result in the production of monocistronic mRNA from which the downstream cistron is translated by conventional cap-dependent, rather than IRES-mediated, initiation.  A later study that documented a variety of unexpected aberrant mRNA species arising from reporter plasmids revealed that splice acceptor sites can mimic both IRES and promoter elements in tests employing such plasmids, further highlighting the need for caution in the interpretation of reporter assay results in the absence of careful RNA analysis. [ 11 ]


IRES sequences are often used in molecular biology to co-express multiple genes under the control of the same promoter, thereby mimicking a polycistronic mRNA. Within the past decades, IRES sequences have been used to develop hundreds of genetically modified rodent animal models. [ 12 ]  The advantage of this technique is that molecular handling is improved. The problem about IRES is that the expression for each subsequent gene is decreased. [ 13 ]


Another viral element to establish polycistronic mRNA in eukaryotes are  2A-peptides . Here, the potential decrease in gene expression and the degree of incomplete separation of proteins is context dependent. [ 14 ]


5-Methyluridine triphosphate  or  m 5 UTP  is one of five  nucleoside triphosphates . [ 1 ]   It is the  ribonucleoside  triphosphate of  thymidine , but the nomenclature with "5-methyluridine" is used because the term  thymidine triphosphate  is used for the  deoxyribonucleoside  by convention. [ 2 ]


N-nucleotides , or nontemplated nucleotides are believed to exist only to create diversity at V(D)J junctions (see  V(D)J recombination ) during lymphocyte development. [ 1 ]  The addition of these nucleotides is aided  by an enzyme called  Terminal deoxynucleotidyl transferase  (TdT)


In  molecular biology , the NAD+ five-prime cap (NAD+ 5' cap) refers to a molecule of  nicotinamide adenine dinucleotide  ( NAD+ ), a nucleoside-containing metabolite, covalently bonded the  5' end  of cellular  mRNA . While the more common  methylated guanosine  ( m7G ) cap is added to  RNA  by a capping complex that associates with  RNA polymerase II  ( RNAP II ), [ 1 ]  the NAD cap is added during transcriptional initiation by the  RNA polymerase  itself, acting as a non-canonical initiating nucleotide (NCIN). [ 2 ]  As such, while m7G capping can only occur in organisms possessing specialized capping complexes, because NAD capping is performed by  RNAP  itself, it is hypothesized to occur in most, if not all, organisms. [ 2 ]


The NAD+ 5' cap has been observed in  bacteria , [ 3 ]  contrary to the long-held belief that prokaryotes lacked  5'-capped   RNA , [ 4 ]  as well as on the  5' cap  of  eukaryotic   mRNA , [ 5 ]  in place of the m7G cap. This modification also potentially allows for selective degradation of RNA]within prokaryotes as different pathways are involved in the degradation of NAD+-capped and uncapped 5′-triphosphate-RNAs.


In  eukaryotic  cells, while the more commonly observed m7G cap promotes the stability of the  mRNA  and supports  translation , [ 6 ]  the NAD+ cap targets the RNA transcript for decay, facilitated by the non-canonical decapping enzyme, DXO. [ 7 ]  Considering the centrality of  NAD  in redox chemistry and  post-translational protein modification , its attachment to RNA represents potentially undiscovered pathways in RNA metabolism and regulation. [ 8 ]


In  prokaryotes , the 5' NAD+ modification is established by bacterial  RNAP  during transcription initiation [ 4 ]  and has been shown to display functions analogous to those of the eukaryotic 5' cap. [ 7 ]  In-vitro transcribed NAD-modified RNA was shown to be more resistant to  RNase E , the main enzyme in the decay pathway of  E. coli . [ 7 ]  NAD-modification further was shown to decelerate RNA processing by RNA pyrophosphohydrolase (RppH), [ 7 ]  which is known to trigger RNase-E-mediated decay through the conversion of 5′-triphosphate-RNA to 5′-monophosphate-RNA. [ 9 ]  Nudc, a  nudix phosphohydrolase , can decap NAD-RNA through  hydrolyzing  NAD(H) into NMN(H) and AMP, [ 10 ]  causing RNase-E-mediated decay, but is inactive against 5′-triphosphate-RNA. [ 7 ]  This 5' modification allows for the selective initiation of degradation for a subset of RNAs as the NAD-capped RNAs are stabilized in the presence of RppH, but are decapped by Nudc, while the 5′-triphosphate-RNAs are susceptible to RppH but not Nudc. [ 11 ]


Next generation sequencing  ( NGS ) of the NAD-RNA conjugates in  E. coli  revealed an abundance of a specific group of small regulatory RNAs (sRNAs) which are known to be involved in stress response systems, as well as  enzymes  involved in  cellular metabolism . [ 12 ]  The small number of RNA transcripts with a NAD cap might allow the cell to selectively degrade these RNAs separate from other pathways. [ 8 ]  Considering that the stress responses are known to affect  NAD+  concentration, [ 13 ]  this finding further supports the possibility of undiscovered pathways linking the energetic state of a cell to mRNA turnover.


NAD capping has also been suggested to recruit specific proteins to the  5' end  of the RNA as NAD is one of the most common  protein ligands . [ 8 ]  NAD-binding pockets are well characterized in many  proteins  and could help the localization of the RNA to an  enzyme  or  receptor . Many NAD-utilizing metabolic enzymes can also bind to  RNA , [ 12 ]  presenting the possibility of unknown  ribonucleoprotein  complexes.


NAD+ 5' capped  RNA  have been found in  yeast , [ 2 ]   humans , [ 4 ]  and  Arabidopsis thaliana . [ 14 ]  In  eukaryotes , the NAD+ cap is removed by non-canonical  decapping  enzymes from the DXO family. [ 15 ]  DeNADing by DXO results in a 5' end monophosphate RNA [ 15 ]  distinct from NudC which results in NMN plus 5′ monophosphate RNA. [ 10 ]  Importantly, DXO is ~6 fold more efficient at decapping NAD+ compared to  m7G , [ 6 ]  suggesting that it selectively degrades NAD-capped RNA rather than the more common m7G cap, similar to NudC.


The  m7G   cap  has been shown to promote translation through recruitment of the initiation complex onto the  mRNA . [ 16 ]  However, the NAD+-cap does not provide a similar function as NAD+-capped and  polyadenylated   mRNA  displayed similar levels of translation  in vitro  to uncapped  mRNA . [ 7 ]  Additionally, the 5' NAD+ cap further promotes decay of the RNA it is attached to, [ 6 ]  NAD+-capped and  polyadenylated   mRNA  were demonstrated  in vitro  to be less stable than mRNAs lacking a  5' cap , [ 7 ]  suggesting that the NAD+ modification is actively facilitating DXO-mediated RNA decay. [ 6 ]


While the relationship between RNA-binding proteins, such as  glyceraldehyde-3-phosphate dehydrogenase  ( GAPDH ), [ 17 ]  and NAD+ concentration is established, the NAD+ cap has been hypothesized to represent a direct link between RNA expression levels and cellular metabolism. [ 6 ]  It is known that energy stresses such as  glucose  deprivation [ 13 ]  and  caloric restriction [ 18 ]  influence NAD+ concentrations and can possibly impact NAD+ capping. Additionally, as low-nutrient conditions can affect  mRNA  stability, [ 19 ]  and seeing as NAD+ caps promote  mRNA  decay, it is possible that the energetic state of a cell could affect NAD+-capping and thus  mRNA  turnover. [ 6 ]  Certain findings, such as the higher abundance of NAD+-capped transcripts in  stationary-phase   bacteria [ 2 ]  as well as  yeast  grown on synthetic media, [ 5 ]  point toward this possibility.


5'-guanosyl-methylene-triphosphate (GDPCP)  and  5'-adenosyl-methylene-triphosphate (ADPCP)  are  analogues  of  guanosine 5'-triphosphate  (GTP) and  adenosine 5'-triphosphate  (ATP), which store  chemical energy  from  metabolism  in the  cell .  Hydrolysis  of  nucleoside triphosphates  (NTPs) such as ATP and GTP yields energy,  inorganic phosphate  (P i  or PP i ), and either NDP or NMP. GDPCP and ADPCP are not subject to hydrolysis under the same conditions as NTPs; it is this property which makes them useful in experiments in  biochemistry  and  molecular biology .


NTPs can be  hydrolyzed  at the  phosphodiester   bonds  between  phosphates , releasing energy and one or more of the three phosphate groups. Additionally, NTPs are inextricable components of some  proteins , where their role may be  structural  and need not involve hydrolysis. In some cases, the presence of an NTP may be required for association of one protein with another, while hydrolysis is necessary for dissociation. GDPCP and ADPCP could be used in such a case, since association can still occur, but hydrolysis-dependent dissociation cannot.


GDPCP was used to examine the  prokaryotic   elongation factor   EF-Tu . EF-Tu is required for the elongation phase of  protein synthesis  ( translation ). EF-Tu requires GTP in order for the  ribosome  to bind it, necessary for recruiting an  aminoacyl-tRNA . The later dissociation of EF-Tu from the ribosome, however, requires that the GTP first be hydrolyzed to GDP and P i . GDPCP was used in place of GTP to differentiate between these two steps: the elongation factor could associate, but without hydrolysis, it was effectively stuck. [ 1 ] [ 2 ]


Nicotinamide adenine dinucleotide  ( NAD ) is a  coenzyme  central to  metabolism . [ 3 ]  Found in all living  cells , NAD is called a dinucleotide because it consists of two  nucleotides  joined through their  phosphate  groups. One nucleotide contains an  adenine   nucleobase  and the other,  nicotinamide . NAD exists in two forms: an  oxidized and reduced  form, abbreviated as  NAD +  and  NADH  (H for  hydrogen ), respectively.


In cellular metabolism, NAD is involved in redox reactions, carrying  electrons  from one reaction to another, so it is found in two forms: NAD +  is an  oxidizing agent , accepting electrons from other molecules and becoming reduced; with H + , this reaction forms NADH, which can be used as a  reducing agent  to donate electrons. These  electron transfer  reactions are the main function of NAD. It is also used in other cellular processes, most notably as a  substrate  of  enzymes  in adding or removing  chemical groups  to or from  proteins , in  posttranslational modifications . Because of the importance of these functions, the enzymes involved in NAD metabolism are targets for  drug discovery .


In organisms, NAD can be synthesized from simple building-blocks ( de novo ) from either  tryptophan  or  aspartic acid , each a case of an  amino acid . Alternatively, more complex components of the coenzymes are taken up from nutritive compounds such as  niacin ; similar compounds are produced by reactions that break down the structure of NAD, providing a  salvage pathway  that recycles them back into their respective active form.


Some NAD is converted into the coenzyme  nicotinamide adenine dinucleotide phosphate  (NADP), whose chemistry largely parallels that of NAD, though its predominant role is as a coenzyme in  anabolic  metabolism.


In the name NAD + , the  superscripted  plus sign indicates the positive  formal charge  on one of its nitrogen atoms.
A biological coenzyme that acts as an electron carrier in enzymatic reactions. NADP is a reducing agent in anabolic reactions like the Calvin cycle and lipid and nucleic acid syntheses. NADP exists in two forms: NADP+, the oxidized form, and NADPH, the reduced form. NADP is similar to nicotinamide adenine dinucleotide (NAD), but NADP has a phosphate group at the C-2′ position of the adenosyl


Nicotinamide adenine dinucleotide consists of two  nucleosides  joined by  pyrophosphate . The nucleosides each contain a  ribose  ring, one with  adenine  attached to the first carbon atom (the  1'  position) ( adenosine diphosphate ribose ) and the other with  nicotinamide  at this position. [ 4 ] [ 5 ]


The compound accepts or donates the equivalent of H − . [ 6 ]  Such reactions (summarized in formula below) involve the removal of two hydrogen atoms from the reactant (R), in the form of a  hydride ion  (H − ), and a  proton  (H + ). The proton is released into solution, while the reductant RH 2  is oxidized and NAD +  reduced to NADH by transfer of the hydride to the nicotinamide ring.


From the hydride electron pair, one electron is attracted to the slightly more electronegative atom of the nicotinamide ring of NAD + , becoming part of the nicotinamide moiety. The second electron and proton atom are transferred to the carbon atom adjacent to the N atom. The  midpoint potential  of the NAD + /NADH redox pair is −0.32  volts , which makes NADH a moderately strong  reducing  agent. [ 7 ]  The reaction is easily reversible, when NADH reduces another molecule and is re-oxidized to NAD + . This means the coenzyme can continuously cycle between the NAD +  and NADH forms without being consumed. [ 5 ]


In appearance, all forms of this coenzyme are white  amorphous  powders that are  hygroscopic  and highly water-soluble. [ 8 ]  The solids are stable if stored dry and in the dark. Solutions of NAD +  are colorless and stable for about a week at 4  °C  and neutral  pH , but decompose rapidly in acidic or alkaline solutions. Upon decomposition, they form products that are  enzyme inhibitors . [ 9 ]


Both NAD +  and NADH strongly absorb  ultraviolet  light because of the adenine. For example, peak absorption of NAD +  is at a  wavelength  of 259  nanometers  (nm), with an  extinction coefficient  of 16,900  M −1 cm −1 . NADH also absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 M −1 cm −1 . [ 10 ]  This difference in the ultraviolet  absorption spectra  between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in  enzyme assays  – by measuring the amount of UV absorption at 340 nm using a  spectrophotometer . [ 10 ]


NAD +  and NADH also differ in their  fluorescence . Freely diffusing NADH in aqueous solution, when excited at the nicotinamide absorbance of ~335 nm (near-UV), fluoresces at 445–460 nm (violet to blue) with a  fluorescence lifetime  of 0.4  nanoseconds , while NAD +  does not fluoresce. [ 11 ] [ 12 ]  The properties of the fluorescence signal changes when NADH binds to  proteins , so these changes can be used to measure  dissociation constants , which are useful in the study of  enzyme kinetics . [ 12 ] [ 13 ]  These changes in fluorescence are also used to measure changes in the redox state of living cells, through  fluorescence microscopy . [ 14 ]


NADH can be converted to NAD+ in a reaction catalysed by copper, which requires hydrogen peroxide. Thus, the supply of NAD+ in cells requires mitochondrial copper(II). [ 15 ] [ 16 ]


In rat liver, the total amount of NAD +  and NADH is approximately 1  μmole  per  gram  of wet weight, about 10 times the concentration of NADP +  and NADPH in the same cells. [ 17 ]  The actual concentration of NAD +  in cell  cytosol  is harder to measure, with recent estimates in animal cells ranging around 0.3  mM , [ 18 ] [ 19 ]  and approximately 1.0 to 2.0 mM in  yeast . [ 20 ]  However, more than 80% of NADH fluorescence in mitochondria is from bound form, so the concentration in solution is much lower. [ 21 ]


NAD +  concentrations are highest in the mitochondria, constituting 40% to 70% of the total cellular NAD + . [ 22 ]  NAD +  in the cytosol is carried into the mitochondrion by a specific  membrane transport protein , since the coenzyme cannot  diffuse  across membranes. [ 23 ]  The intracellular  half-life  of NAD +  was claimed to be between 1–2 hours by one review, [ 24 ]  whereas another review gave varying estimates based on compartment: intracellular 1–4 hours, cytoplasmic 2 hours, and mitochondrial 4–6 hours. [ 25 ]


The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD + /NADH ratio. This ratio is an important component of what is called the  redox state  of a cell, a measurement that reflects both the metabolic activities and the health of cells. [ 26 ]  The effects of the NAD + /NADH ratio are complex, controlling the activity of several key enzymes, including  glyceraldehyde 3-phosphate dehydrogenase  and  pyruvate dehydrogenase . In healthy mammalian tissues, estimates of the ratio of free NAD +  to NADH in the cytoplasm typically lie around 700:1; the ratio is thus favorable for oxidative reactions. [ 27 ] [ 28 ]  The ratio of total NAD + /NADH is much lower, with estimates ranging from 3–10 in mammals. [ 29 ]  In contrast, the  NADP + /NADPH  ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. [ 30 ]  These different ratios are key to the different metabolic roles of NADH and NADPH.


NAD +  is synthesized through two metabolic pathways. It is produced either in a  de novo  pathway from amino acids or in salvage pathways by recycling preformed components such as  nicotinamide  back to NAD + . Although most tissues synthesize NAD +  by the salvage pathway in mammals, much more  de novo  synthesis occurs in the liver from tryptophan, and in the kidney and  macrophages  from  nicotinic acid . [ 31 ]


Most organisms synthesize NAD +  from simple components. [ 6 ]  The specific set of reactions differs among organisms, but a common feature is the generation of  quinolinic acid  (QA) from an amino acid – either  tryptophan  (Trp) in animals and some bacteria, or  aspartic acid  (Asp) in some bacteria and plants. [ 32 ] [ 33 ]  The quinolinic acid is converted to nicotinic acid mononucleotide (NaMN) by transfer of a phosphoribose moiety. An adenylate moiety is then transferred to form nicotinic acid adenine dinucleotide (NaAD). Finally, the nicotinic acid moiety in NaAD is  amidated  to a nicotinamide (Nam) moiety, forming nicotinamide adenine dinucleotide. [ 6 ]


In a further step, some NAD +  is converted into NADP +  by  NAD +  kinase , which  phosphorylates  NAD + . [ 34 ]  In most organisms, this enzyme uses  adenosine triphosphate  (ATP) as the source of the phosphate group, although several bacteria such as  Mycobacterium tuberculosis  and a hyperthermophilic  archaeon   Pyrococcus horikoshii , use inorganic  polyphosphate  as an alternative phosphoryl donor. [ 35 ] [ 36 ]


Despite the presence of the  de novo  pathway, the salvage reactions are essential in humans; a lack of  niacin  in the diet causes the  vitamin deficiency  disease  pellagra . [ 37 ]  This high requirement for NAD +  results from the constant consumption of the coenzyme in reactions such as posttranslational modifications, since the cycling of NAD +  between oxidized and reduced forms in redox reactions does not change the overall levels of the coenzyme. [ 6 ]
The major source of NAD +  in mammals is the salvage pathway which recycles the  nicotinamide  produced by enzymes utilizing NAD + . [ 38 ]  The first step, and the rate-limiting enzyme in the salvage pathway is  nicotinamide phosphoribosyltransferase  (NAMPT), which produces  nicotinamide mononucleotide  (NMN). [ 38 ]  NMN is the immediate precursor to NAD+ in the salvage pathway. [ 39 ]


Besides assembling NAD +   de novo  from simple amino acid precursors, cells also salvage preformed compounds containing a pyridine base. The three vitamin precursors used in these salvage metabolic pathways are nicotinic acid (NA), nicotinamide (Nam) and  nicotinamide riboside  (NR). [ 6 ]  These compounds can be taken up from the diet and are termed vitamin B 3  or  niacin . However, these compounds are also produced within cells and by digestion of cellular NAD + . Some of the enzymes involved in these salvage pathways appear to be concentrated in the  cell nucleus , which may compensate for the high level of reactions that consume NAD +  in this  organelle . [ 40 ]  There are some reports that mammalian cells can take up extracellular NAD +  from their surroundings, [ 41 ]  and both nicotinamide and nicotinamide riboside can be absorbed from the gut. [ 42 ]


The salvage pathways used in  microorganisms  differ from those of  mammals . [ 43 ]  Some pathogens, such as the yeast  Candida glabrata  and the bacterium  Haemophilus influenzae  are NAD +   auxotrophs  – they cannot synthesize NAD +  – but possess salvage pathways and thus are dependent on external sources of NAD +  or its precursors. [ 44 ] [ 45 ]  Even more surprising is the intracellular  pathogen   Chlamydia trachomatis , which lacks recognizable candidates for any genes involved in the biosynthesis or salvage of both NAD +  and NADP + , and must acquire these coenzymes from its  host . [ 46 ]


Nicotinamide adenine dinucleotide has several essential roles in  metabolism . It acts as a  coenzyme  in  redox  reactions, as a donor of ADP-ribose moieties in  ADP-ribosylation  reactions, as a precursor of the  second messenger  molecule  cyclic ADP-ribose , as well as acting as a substrate for bacterial  DNA ligases  and a group of enzymes called  sirtuins  that use NAD +  to remove  acetyl  groups from proteins. In addition to these metabolic functions, NAD +  emerges as an adenine nucleotide that can be released from cells spontaneously and by regulated mechanisms, [ 48 ] [ 49 ]  and can therefore have important  extracellular  roles. [ 49 ]


The main role of NAD +  in metabolism is the transfer of electrons from one molecule to another. Reactions of this type are catalyzed by a large group of enzymes called  oxidoreductases . The correct names for these enzymes contain the names of both their substrates: for example  NADH-ubiquinone oxidoreductase  catalyzes the oxidation of NADH by  coenzyme Q . [ 50 ]  However, these enzymes are also referred to as  dehydrogenases  or  reductases , with NADH-ubiquinone oxidoreductase commonly being called  NADH dehydrogenase  or sometimes  coenzyme Q reductase . [ 51 ]


There are many different superfamilies of enzymes that bind NAD +  / NADH. One of the most common superfamilies includes a  structural motif  known as the  Rossmann fold . [ 52 ] [ 53 ]  The motif is named after  Michael Rossmann , who was the first scientist to notice how common this structure is within nucleotide-binding proteins. [ 54 ]


An example of a NAD-binding bacterial enzyme involved in  amino acid  metabolism that does not have the Rossmann fold is found in  Pseudomonas syringae  pv. tomato ( PDB :  2CWH ;  InterPro :  IPR003767 ). [ 55 ]


When bound in the active site of an oxidoreductase, the nicotinamide ring of the coenzyme is positioned so that it can accept a hydride from the other substrate. Depending on the enzyme, the hydride donor is positioned either "above" or "below" the plane of the planar C4 carbon, as defined in the figure. Class A oxidoreductases transfer the atom from above; class B enzymes transfer it from below. Since the C4 carbon that accepts the hydrogen is  prochiral , this can be exploited in  enzyme kinetics  to give information about the enzyme's mechanism. This is done by mixing an enzyme with a substrate that has  deuterium  atoms substituted for the hydrogens, so the enzyme will reduce NAD +  by transferring deuterium rather than hydrogen. In this case, an enzyme can produce one of two  stereoisomers  of NADH. [ 56 ]


Despite the similarity in how proteins bind the two coenzymes, enzymes almost always show a high level of specificity for either NAD +  or NADP + . [ 57 ]  This specificity reflects the distinct metabolic roles of the respective coenzymes, and is the result of distinct sets of  amino acid  residues in the two types of coenzyme-binding pocket. For instance, in the active site of NADP-dependent enzymes, an  ionic bond  is formed between a basic amino acid side-chain and the acidic phosphate group of NADP + . On the converse, in NAD-dependent enzymes the charge in this pocket is reversed, preventing NADP +  from binding. However, there are a few exceptions to this general rule, and enzymes such as  aldose reductase ,  glucose-6-phosphate dehydrogenase , and  methylenetetrahydrofolate reductase  can use both coenzymes in some species. [ 58 ]


The redox reactions catalyzed by oxidoreductases are vital in all parts of metabolism, but one particularly important area where these reactions occur is in the release of energy from nutrients. Here, reduced compounds such as  glucose  and  fatty acids  are oxidized, thereby releasing energy. This energy is transferred to NAD +  by reduction to NADH, as part of  beta oxidation ,  glycolysis , and the  citric acid cycle . In  eukaryotes  the electrons carried by the NADH that is produced in the  cytoplasm  are transferred into the  mitochondrion  (to reduce mitochondrial NAD + ) by  mitochondrial shuttles , such as the  malate-aspartate shuttle . [ 59 ]  The mitochondrial NADH is then oxidized in turn by the  electron transport chain , which pumps protons across a membrane and generates ATP through  oxidative phosphorylation . [ 60 ]  These shuttle systems also have the same transport function in  chloroplasts . [ 61 ]


Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions, the cell maintains significant concentrations of both NAD +  and NADH, with the high NAD + /NADH ratio allowing this coenzyme to act as both an oxidizing and a reducing agent. [ 62 ]  In contrast, the main function of NADPH is as a reducing agent in  anabolism , with this coenzyme being involved in pathways such as  fatty acid synthesis  and  photosynthesis . Since NADPH is needed to drive redox reactions as a strong reducing agent, the NADP + /NADPH ratio is kept very low. [ 62 ]


Although it is important in catabolism, NADH is also used in anabolic reactions, such as  gluconeogenesis . [ 63 ]  This need for NADH in anabolism poses a problem for prokaryotes growing on nutrients that release only a small amount of energy. For example,  nitrifying  bacteria such as  Nitrobacter  oxidize nitrite to nitrate, which releases sufficient energy to pump protons and generate ATP, but not enough to produce NADH directly. [ 64 ]  As NADH is still needed for anabolic reactions, these bacteria use a  nitrite oxidoreductase  to produce enough  proton-motive force  to run part of the electron transport chain in reverse, generating NADH. [ 65 ]


The coenzyme NAD +  is also consumed in ADP-ribose transfer reactions. For example, enzymes called  ADP-ribosyltransferases  add the ADP-ribose moiety of this molecule to proteins, in a  posttranslational modification  called  ADP-ribosylation . [ 66 ]  ADP-ribosylation involves either the addition of a single ADP-ribose moiety, in  mono-ADP-ribosylation , or the transferral of ADP-ribose to proteins in long branched chains, which is called  poly(ADP-ribosyl)ation . [ 67 ]  Mono-ADP-ribosylation was first identified as the mechanism of a group of bacterial  toxins , notably  cholera toxin , but it is also involved in normal  cell signaling . [ 68 ] [ 69 ]  Poly(ADP-ribosyl)ation is carried out by the  poly(ADP-ribose) polymerases . [ 67 ] [ 70 ]  The poly(ADP-ribose) structure is involved in the regulation of several cellular events and is most important in the  cell nucleus , in processes such as  DNA repair  and  telomere  maintenance. [ 70 ]  In addition to these functions within the cell, a group of  extracellular  ADP-ribosyltransferases has recently been discovered, but their functions remain obscure. [ 71 ]
NAD +  may also be added onto cellular  RNA  as a 5'-terminal modification. [ 72 ]


Another function of this coenzyme in cell signaling is as a precursor of  cyclic ADP-ribose , which is produced from NAD +  by ADP-ribosyl cyclases, as part of a  second messenger system . [ 73 ]  This molecule acts in  calcium signaling  by releasing calcium from intracellular stores. [ 74 ]  It does this by binding to and opening a class of calcium channels called  ryanodine receptors , which are located in the membranes of  organelles , such as the  endoplasmic reticulum , and inducing the activation of the  transcription factor   NAFC3 [ 75 ]


NAD +  is also consumed by different NAD+-consuming enzymes, such as  CD38 ,  CD157 ,  PARPs  and the NAD-dependent  deacetylases  ( sirtuins ,such as  Sir2 . [ 76 ] ). [ 77 ]  These enzymes act by transferring an  acetyl  group from their substrate protein to the ADP-ribose moiety of NAD + ; this cleaves the coenzyme and releases nicotinamide and O-acetyl-ADP-ribose. The sirtuins mainly seem to be involved in regulating  transcription  through deacetylating histones and altering  nucleosome  structure. [ 78 ]  However, non-histone proteins can be deacetylated by sirtuins as well. These activities of sirtuins are particularly interesting because of their importance in the regulation of  aging . [ 79 ] [ 80 ]


Other NAD-dependent enzymes include bacterial  DNA ligases , which join two DNA ends by using NAD +  as a substrate to donate an  adenosine monophosphate  (AMP) moiety to the 5' phosphate of one DNA end. This intermediate is then attacked by the 3' hydroxyl group of the other DNA end, forming a new  phosphodiester bond . [ 81 ]  This contrasts with  eukaryotic  DNA ligases, which use ATP to form the DNA-AMP intermediate. [ 82 ]


Li et al. have found that NAD +  directly regulates protein-protein interactions. [ 83 ]  They also show that one of the causes of age-related decline in DNA repair may be increased binding of the protein  DBC1  (Deleted in Breast Cancer 1) to  PARP1  (poly[ADP–ribose] polymerase 1) as NAD +  levels decline during aging. [ 83 ]   The decline in cellular concentrations of NAD +   during aging likely contributes to the  aging  process and to the  pathogenesis  of the chronic diseases of aging. [ 84 ]   Thus, the modulation of NAD +  may protect against cancer, radiation, and aging. [ 83 ]


In recent years, NAD +  has also been recognized as an  extracellular  signaling molecule involved in cell-to-cell communication. [ 49 ] [ 85 ] [ 86 ]  NAD +  is released from  neurons  in  blood vessels , [ 48 ]   urinary bladder , [ 48 ] [ 87 ]   large intestine , [ 88 ] [ 89 ]  from neurosecretory cells, [ 90 ]  and from brain  synaptosomes , [ 91 ]  and is proposed to be a novel  neurotransmitter  that transmits information from  nerves  to effector cells in  smooth muscle  organs. [ 88 ] [ 89 ]  In plants, the extracellular nicotinamide adenine dinucleotide induces resistance to pathogen infection and the first extracellular NAD receptor has been identified. [ 92 ]  Further studies are needed to determine the underlying mechanisms of its extracellular actions and their importance for human health and life processes in other organisms.


The enzymes that make and use NAD +  and NADH are important in both  pharmacology  and the research into future treatments for disease. [ 93 ]   Drug design  and drug development exploits NAD +  in three ways: as a direct target of drugs, by designing  enzyme inhibitors  or activators based on its structure that change the activity of NAD-dependent enzymes, and by trying to inhibit NAD +  biosynthesis. [ 94 ]


Because cancer cells utilize increased  glycolysis , and because NAD enhances glycolysis, nicotinamide phosphoribosyltransferase (NAD salvage pathway) is often amplified in cancer cells. [ 95 ] [ 96 ]


It has been studied for its potential use in the therapy of  neurodegenerative diseases  such as  Alzheimer's  and  Parkinson's disease  as well as  multiple sclerosis . [ 6 ] [ 80 ] [ 97 ] [ 77 ]  A placebo-controlled clinical trial of NADH (which excluded NADH precursors) in people with Parkinson's failed to show any effect. [ 98 ]


NAD +  is also a direct  target  of the drug  isoniazid , which is used in the treatment of  tuberculosis , an infection caused by  Mycobacterium tuberculosis . Isoniazid is a  prodrug  and once it has entered the bacteria, it is activated by a  peroxidase  enzyme, which oxidizes the compound into a  free radical  form. [ 99 ]  This radical then reacts with NADH, to produce adducts that are very potent inhibitors of the enzymes  enoyl-acyl carrier protein reductase , [ 100 ]  and  dihydrofolate reductase . [ 101 ]


Since many oxidoreductases use NAD +  and NADH as substrates, and bind them using a highly conserved structural motif, the idea that inhibitors based on NAD +  could be specific to one enzyme is surprising. [ 102 ]  However, this can be possible: for example, inhibitors based on the compounds  mycophenolic acid  and  tiazofurin  inhibit  IMP dehydrogenase  at the NAD +  binding site. Because of the importance of this enzyme in  purine metabolism , these compounds may be useful as anti-cancer, anti-viral, or  immunosuppressive drugs . [ 102 ] [ 103 ]  Other drugs are not enzyme inhibitors, but instead activate enzymes involved in NAD +  metabolism.  Sirtuins  are a particularly interesting target for such drugs, since activation of these NAD-dependent deacetylases extends lifespan in some animal models. [ 104 ]  Compounds such as  resveratrol  increase the activity of these enzymes, which may be important in their ability to delay aging in both vertebrate, [ 105 ]  and invertebrate  model organisms . [ 106 ] [ 107 ]  In one experiment, mice given NAD for one week had improved nuclear-mitochrondrial communication. [ 108 ]


Because of the differences in the  metabolic pathways  of NAD +  biosynthesis between organisms, such as between bacteria and humans, this area of metabolism is a promising area for the development of new  antibiotics . [ 109 ] [ 110 ]  For example, the enzyme  nicotinamidase , which converts nicotinamide to nicotinic acid, is a target for drug design, as this enzyme is absent in humans but present in yeast and bacteria. [ 43 ]


In bacteriology, NAD, sometimes referred to factor V, is used as a supplement to culture media for some  fastidious  bacteria. [ 111 ]


The coenzyme NAD +  was first discovered by the British biochemists  Arthur Harden  and  William John Young  in 1906. [ 112 ]  They noticed that adding boiled and filtered  yeast  extract greatly accelerated  alcoholic fermentation  in unboiled yeast extracts. They called the unidentified factor responsible for this effect a  coferment . Through a long and difficult purification from yeast extracts, this heat-stable factor was identified as a  nucleotide  sugar phosphate by  Hans von Euler-Chelpin . [ 113 ]  In 1936, the German scientist  Otto Heinrich Warburg  showed the function of the nucleotide coenzyme in hydride transfer and identified the nicotinamide portion as the site of redox reactions. [ 114 ]


Vitamin precursors of NAD +  were first identified in 1938, when  Conrad Elvehjem  showed that liver has an "anti-black tongue" activity in the form of nicotinamide. [ 115 ]  Then, in 1939, he provided the first strong evidence that niacin is used to synthesize NAD + . [ 116 ]  In the early 1940s,  Arthur Kornberg  was the first to detect an enzyme in the biosynthetic pathway. [ 117 ]  In 1949, the American biochemists Morris Friedkin and  Albert L. Lehninger  proved that NADH linked metabolic pathways such as the  citric acid cycle  with the synthesis of ATP in oxidative phosphorylation. [ 118 ]  In 1958, Jack Preiss and Philip Handler discovered the intermediates and enzymes involved in the biosynthesis of NAD + ; [ 119 ] [ 120 ]  salvage synthesis from nicotinic acid is termed the Preiss-Handler pathway. In 2004,  Charles Brenner  and co-workers uncovered the  nicotinamide riboside  kinase pathway to NAD + . [ 121 ]


The non-redox roles of NAD(P) were discovered later. [ 5 ]  The first to be identified was the use of NAD +  as the ADP-ribose donor in ADP-ribosylation reactions, observed in the early 1960s. [ 122 ]  Studies in the 1980s and 1990s revealed the activities of NAD +  and NADP +  metabolites in cell signaling – such as the action of  cyclic ADP-ribose , which was discovered in 1987. [ 123 ]


The metabolism of NAD +  remained an area of intense research into the 21st century, with interest heightened after the discovery of the NAD + -dependent protein deacetylases called  sirtuins  in 2000, by Shin-ichiro Imai and coworkers in the laboratory of  Leonard P. Guarente . [ 124 ]  In 2009 Imai proposed the "NAD World" hypothesis that key regulators of aging and longevity in mammals are  sirtuin 1  and the primary NAD +  synthesizing enzyme  nicotinamide phosphoribosyltransferase  (NAMPT). [ 125 ]  In 2016 Imai expanded his hypothesis to "NAD World 2.0", which postulates that extracellular NAMPT from  adipose tissue  maintains NAD +  in the  hypothalamus  (the control center) in conjunction with  myokines  from  skeletal muscle  cells. [ 126 ]  In 2018, Napa Therapeutics was formed to develop drugs against a novel aging-related target based on the research in NAD metabolism conducted in the lab of  Eric Verdin . [ 127 ]


Nicotinamide adenine dinucleotide phosphate , abbreviated  NADP [ 1 ] [ 2 ]  or, in older notation,  TPN  (triphosphopyridine nucleotide), is a  cofactor  used in  anabolic reactions , such as the  Calvin cycle  and  lipid  and  nucleic acid  syntheses, which require  NADPH  as a  reducing agent  ('hydrogen source'). NADPH is the  reduced  form, whereas NADP +  is the  oxidized  form. NADP +  is used by all forms of cellular life. NADP +  is essential for life because it is needed for cellular respiration. [ 3 ]


NADP +  differs from  NAD +  by the presence of an additional  phosphate group  on the 2' position of the  ribose  ring that carries the  adenine   moiety . This extra phosphate is added by  NAD +  kinase  and removed by NADP +  phosphatase. [ 4 ]


In general, NADP +  is synthesized before NADPH is. Such a reaction usually starts with  NAD +  from either the de-novo or the salvage pathway, with  NAD +  kinase  adding the extra phosphate group.  ADP-ribosyl cyclase  allows for synthesis from  nicotinamide  in the salvage pathway, and NADP +  phosphatase can convert NADPH back to NADH to maintain a balance. [ 3 ]  Some forms of the NAD +  kinase, notably the one in mitochondria, can also accept NADH to turn it directly into NADPH. [ 5 ] [ 6 ]  The prokaryotic pathway is less well understood, but with all the similar proteins the process should work in a similar way. [ 3 ]


NADPH is produced from NADP + . The major source of NADPH in animals and other non-photosynthetic organisms is the  pentose phosphate pathway , by  glucose-6-phosphate dehydrogenase  (G6PDH) in the first step. The pentose phosphate pathway also produces pentose, another important part of NAD(P)H, from glucose. Some bacteria also use G6PDH for the  Entner–Doudoroff pathway , but NADPH production remains the same. [ 3 ]


Ferredoxin–NADP +  reductase , present in all domains of life, is a major source of NADPH in photosynthetic organisms including plants and cyanobacteria. It appears in the last step of the electron chain of the  light reactions  of  photosynthesis . It is used as reducing power for the biosynthetic reactions in the  Calvin cycle  to assimilate carbon dioxide and help turn the carbon dioxide into glucose. It has functions in accepting electrons in other non-photosynthetic pathways as well: it is needed in the reduction of nitrate into ammonia for plant assimilation in  nitrogen cycle  and in the production of oils. [ 3 ]


There are several other lesser-known mechanisms of generating NADPH, all of which depend on the presence of  mitochondria  in eukaryotes. The key enzymes in these carbon-metabolism-related processes are NADP-linked isoforms of  malic enzyme ,  isocitrate dehydrogenase  (IDH), and  glutamate dehydrogenase . In these reactions, NADP +  acts like NAD +  in other enzymes as an oxidizing agent. [ 7 ]  The isocitrate dehydrogenase mechanism appears to be the major source of NADPH in fat and possibly also liver cells. [ 8 ]  These processes are also found in bacteria. Bacteria can also use a NADP-dependent  glyceraldehyde 3-phosphate dehydrogenase  for the same purpose. Like the pentose phosphate pathway, these pathways are related to parts of  glycolysis . [ 3 ]  Another carbon metabolism-related pathway involved in the generation of NADPH is the mitochondrial folate cycle, which uses principally serine as a source of one-carbon units to sustain nucleotide synthesis and redox homeostasis in mitochondria. Mitochondrial folate cycle has been recently suggested as the principal contributor to NADPH generation in mitochondria of cancer cells. [ 9 ]


NADPH can also be generated through pathways unrelated to carbon metabolism. The ferredoxin reductase is such an example.  Nicotinamide nucleotide transhydrogenase  transfers the hydrogen between NAD(P)H and NAD(P) + , and is found in eukaryotic mitochondria and many bacteria. There are versions that depend on a  proton gradient  to work and ones that do not. Some anaerobic organisms use  NADP + -linked hydrogenase , ripping a hydride from hydrogen gas to produce a proton and NADPH. [ 3 ]


Like  NADH , NADPH is  fluorescent . NADPH in aqueous solution excited at the nicotinamide absorbance of ~335 nm (near UV) has a fluorescence emission which peaks at 445-460 nm (violet to blue). NADP +  has no appreciable fluorescence. [ 10 ]


NADPH provides the reducing agents, usually hydrogen atoms, for biosynthetic reactions and the  oxidation-reduction  involved in protecting against the toxicity of  reactive oxygen species  (ROS), allowing the regeneration of  glutathione  (GSH). [ 11 ]  NADPH is also used for  anabolic  pathways, such as  cholesterol synthesis , steroid synthesis, [ 12 ]  ascorbic acid synthesis, [ 12 ]  xylitol synthesis, [ 12 ]  cytosolic fatty acid synthesis [ 12 ]  and microsomal  fatty acid chain elongation .


The NADPH system is also responsible for generating free radicals in immune cells by  NADPH oxidase . These radicals are used to destroy pathogens in a process termed the  respiratory burst . [ 13 ]
It is the source of reducing equivalents for  cytochrome P450   hydroxylation  of  aromatic compounds ,  steroids ,  alcohols , and  drugs .


NADH and NADPH are very stable in basic solutions, but NAD +  and NADP +  are degraded in basic solutions into a fluorescent product that can be used conveniently for quantitation. Conversely, NADPH and NADH are degraded by acidic solutions while NAD + /NADP +  are fairly stable to acid. [ 14 ] [ 15 ]


Many enzymes that bind NADP share a common super-secondary structure named named the "Rossmann fold". The initial beta-alpha-beta (βαβ) fold is the most conserved segment of the Rossmann folds. This segment is in contact with the ADP portion of NADP. Therefore, it is also called an "ADP-binding βαβ fold". [ 16 ]


In 2018 and 2019, the first two reports of enzymes that catalyze the removal of the 2' phosphate of NADP(H) in eukaryotes emerged. First the  cytoplasmic  protein MESH1 ( Q8N4P3 ), [ 19 ]  then the  mitochondrial  protein  nocturnin [ 20 ]  were reported. Of note, the structures and NADPH binding of MESH1 ( 5VXA ) and nocturnin ( 6NF0 ) are not related.


Nicotinamide mononucleotide  (" NMN " and " β-NMN ") is a  nucleotide  derived from  ribose ,  nicotinamide ,  nicotinamide riboside  and  niacin . [ 1 ]  In humans, several enzymes use NMN to generate  nicotinamide adenine dinucleotide  (NADH). [ 1 ]  In mice, it has been proposed that NMN is absorbed via the small intestine within 10 minutes of oral uptake and converted to nicotinamide adenine dinucleotide ( NAD+ ) through the  Slc12a8  transporter. [ 2 ]  However, this observation has been challenged, [ 3 ]  and the matter remains unsettled. [ 4 ]


Because NADH is a  cofactor  for processes inside  mitochondria , for  sirtuins  and  PARP , NMN has been studied in  animal models  as a potential neuroprotective and  anti-aging  agent. [ 5 ] [ 6 ]  The reversal of aging at the cellular level by inhibiting  mitochondrial decay  in presence of increased levels of NAD+ makes it popular among anti-aging products. [ 7 ]  Dietary supplement companies have  aggressively marketed  NMN products, claiming those benefits. [ 8 ]  However, no human studies to date have properly proven its anti-aging effects with proposed health benefits only suggested through research done  in vitro  or through  animal models . [ 9 ]  Single-dose administration of up to 500 mg was shown safe in men in a study at  Keio University . [ 10 ]  One 2021 clinical trial found that NMN improved muscular insulin sensitivity in prediabetic women, [ 11 ]  while another found that it improved aerobic capacity in amateur runners. [ 12 ]  A 2023 clinical trial showed that NMN improves performance on a six-minute walking test and a subjective general health assessment. [ 13 ]


NMN is vulnerable to extracellular degradation by  CD38  enzyme, [ 14 ]  which can be inhibited by compounds such as  CD38-IN-78c . [ 15 ]


NMN is found in fruits and vegetables such as  edamame ,  broccoli ,  cabbage ,  cucumber  and  avocado  at a concentration of about 1 mg per 100g, [ 16 ] [ 17 ] [ 18 ]  making these natural sources impractical to acquire the quantities needed to accomplish the dosing currently being investigated for NMN as a pharmaceutical.


Production of nicotinamide mononucleotide has been redacted since the latter half of 2022 by the  FDA  because it is under investigation as a pharmaceutical drug. [ 19 ] [ 20 ]


The synthesizing enzymes and consumption enzymes of NMN also exhibit tissue specificity: NMN is widely distributed in tissues and organs throughout the body and has been present in various cells since embryonic development. [ 20 ]


NMN is a precursor for  NAD +  biosynthesis, and NMN dietary supplementation has been demonstrated to increase NAD +  concentration and thus has the potential to mitigate aging-related disorders such as  oxidative stress ,  DNA damage ,  neurodegeneration  and  inflammatory responses . [ 21 ]  The potential benefits and risks of NMN supplementation, as of 2023, are currently under investigation. [ 21 ]


Certain enzymes are sensitive to the intracellular NMN/ NAD +  ratio, such as  SARM1 , [ 22 ]  a protein responsible for initiating cellular degeneration pathways such as  MAP kinase  and inducing  axonal  loss and  neuronal death . [ 23 ] [ 24 ]   NMNAT  is an enzyme with neurorescuing properties that functions to deplete NMN and produce NAD + , attenuating SARM1 activity and aiding neuronal survival  in-vitro , [ 25 ] [ 26 ]  an effect that is reversed by applying exogenous NMN which promptly resumed axon destruction. [ 23 ]  The similar molecule nicotinic acid mononucleotide (NaMN) opposes the activating effect of NMN on  SARM1 , and is a neuroprotector. [ 27 ]


Nicotinic acid adenine dinucleotide phosphate  ( NAADP ) is a Ca 2+ -mobilizing second messenger synthesised in response to extracellular stimuli. Like its mechanistic cousins,  IP 3  and cyclic adenosine diphosphoribose ( Cyclic ADP-ribose ), NAADP binds to and opens Ca 2+  channels on intracellular organelles, thereby increasing the intracellular Ca 2+  concentration which, in turn, modulates sundry cellular processes (see  Calcium signalling ). Structurally, it is a dinucleotide that only differs from the house-keeping enzyme cofactor,  NADP  by a hydroxyl group (replacing the nicotinamide amino group) and yet this minor modification converts it into the most potent Ca 2+ -mobilizing second messenger yet described. NAADP acts across phyla from  plants  to  humans .


In their landmark 1987 paper, [ 1 ]  Hon Cheung Lee and colleagues discovered not one but two Ca 2+ -mobilizing second messengers,  cADPR  and NAADP from the effects of nucleotides on Ca 2+  release in sea urchin egg homogenates. It turns out that NAADP was a contaminant in commercial sources of  NADP , but it was not until 1995 that its structure was solved. [ 2 ]  The first demonstration that NAADP could act in mammalian cells (pancreas) came four years later. [ 3 ]  Subsequently, NAADP has been detected in sources as diverse as human sperm, red and white blood cells, liver, and pancreas, to name but a few. [ 4 ]


The first demonstration that NAADP levels increase in response to an extracellular stimulus arose from studying sea urchin  fertilization  (NAADP changed in both the eggs and sperm upon contact). [ 5 ]  Subsequently, other cell types have followed suit, as exemplified by the pancreas (acinar and beta cells), T-cells, and smooth muscle. Levels increase very rapidly — and possibly precede the increase in the other messengers IP 3  and cADPR [ 6 ] — but can be very transient (spiking and returning to basal levels within seconds). The transduction mechanisms that couple cell stimuli to such NAADP increases are ill-defined, with some suggestions of  cyclic AMP [ 7 ]  or cytosolic Ca 2+  itself [ 8 ]  stimulating synthesis.