Nicotinamide Mononucleotide (NMN) Adenylyltransferase (NMNAT)

Nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) is a class of enzymes that is a member of the nucleotidyl-adenylyltransferases, which are a family of proteins indispensable for cellular homeostasis (1). A transferase is an enzyme that transfers a chemical group, like a methyl group or a glycosyl group, from one compound (a donor) to another compound (an acceptor). In the case of nucleotidyltransferases, the transfer that occurs is specifically of the phosphorus-containing nucleotide groups.

In adenylyltransferase it is the adenine base on a phosphorylated nucleotide.

NMNATs are classically known for their enzymatic function of catalyzing NAD+ synthesis (1). Due to the importance of nicotinamide adenine dinucleotide (NAD) in all cells as a coenzyme and its involvement in hundreds of metabolic oxidation and reduction reactions, NMNAT is seen as a highly conserved enzyme throughout the evolution of life from archaea, while divergent from human isoforms is very similar to bacteria NMNAT, prokaryotes, and eukaryotes.

There are three different NMNAT's that have been recognized, NMNAT-1 (homohexamer, figure 1) is ubiquitous located in the nucleus of cells, NMNAT-2 (homodimer) in the cytoplasm expressed predominately in the brain, and NMNAT-3 (homotetramer) in the mitochondria and cytosol expressed predominately in the liver, heart, skeletal muscle, and red blood cells of vertebrates (invertebrates have only one isoform of NMNAT) (2, 3).

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Because of the involvement of NMNATs in the pathways of metabolism, and in the case of vertebrates, neuron or axon conservation and repair, there are a multitude of medical uses currently employed or still in the process of being developed that use this enzyme.

NMNAT's catalyze the metabolic conversion of potent antitumor drugs to their active forms and due to the low activity in tumor cells can be used as a source for therapeutic targeting for cancers and other diseases. Further studies are required to determine the exact role NMNAT-2 provides versus NMNAT-1 and NMNAT-3 for neurogenerative conditions including motoneuron disease, Parkinson's disease, glaucoma, and myelin-related axon loss in order to develop new treatment options (2, 5). NMNAT is found in every living organism that uses NAD biosynthetic pathways, including bacteria, plants, and animals.

Although there are three forms of NMNAT recognized, the earliest forms of NMNAT differ quite drastically from the forms we see in more complex organisms. Archaeal NMNAT more closely resembles the forms we see in bacteria. The most common organisms studied that utilize NMNATs are Homo sapiens (3 isoforms, NMNAT-1, NMNAT-2, & NMNAT-3), Drosophila melanogaster, Escherichia coli, Mus musculus, and Methanothermobacyer thermautotrophicus. More research is currently underway to determine the mechanisms, structure and purpose of this enzyme in additional species.

Much of the research on the genetic and structural level, as in the amino acid composition in the active protein in organisms is currently in progress, so many of the species are currently speculated versus having the x-ray crystallography structure of the enzyme(s). The amino acid sequence is highly conserved (> 95%) throughout the species that have been isolated and mapped out, which suggests that NMNATs are essential for all forms of life to survive (figure 7; t-coffee). NMNAT (2.7.7.1) plays an integral role in the NAD biosynthetic pathway (figure 5, 6). The structure of NMNATs varies with the isoform and no x-ray crystallography structure of isoform 2 has been developed at the present time. One interesting feature of NMNAT is the strong conservation of the nucleotide HXGH sequence motif (9). The quaternary structure of NMNAT consists of a homohexamer, which is seen as a trimer of dimers, or three units consisting of two subunits each.

There are two major subunits, the first is related by a dyad axis and the second is related by the threefold axis, both consist of associated protomers along each of the axes. There are many interactions that participate in the dimer stabilization and specifically at the dyad axis there are a Pro69"Ile70"Pro71"Ile72 motif, hydrogen bonding, hydrophobic interactions, a salt bridge between the glutamate and arginine residues, and dipoles between the helices which all contribute to the dimer stabilization (9).

In the case of the triad axis, there are hydrogen bonds and a salt bridge formed by glutamate and arginine residues (9, 10). NMNAT can be viewed as a 60 … long and 50 … wide globular homohexamer that has at its center an 11 … solvent channel crossing the entire hexamer running along the threefold axis (9, 10). The catalytic sites face the solvent channel and are located in a deep clefts within each subunit that extend from the top of the hexamer to the trimer"trimer interface, which can be seen in figures 2 and 3 by the blue, red, grey, and orange molecules. Positively charged amino acid residues surround the entrance of the channel to interact with the ATP (a very negatively charged molecule), and negatively charged aspartate and glutamate residues are located at the trimer"trimer interface (9, 10).

The ATP-binding site in a deep cleft is between the first and the fourth parallel І strands and Mg2+ ligands are provided by ±-, І- and і-phosphate oxygen atoms which are required for the ATP molecule to associate in the deep clefts of each active site. Each binding site, or active site, that face the channel are highly solvent accessible, which is why stabilization of the ATP is so important for function and contribute to this enzyme's nucleotide specificity. The three phosphate groups of the ATP molecule are stabilized by the interaction of several protein residues near the adenosine, including the і-phosphate which forms a salt bridge with arginine residues, and hydrogen bonds with the hydroxyl group of the threonine residue.

The І-phosphate interacts with the NH2 group of the arginine residue and a mainchain serine residue, and because the І-phosphate is centered at the N-terminus of one of the helices the dipole also contributes to the stabilization of ATP in the active sites (9, 10). Nicotinamide mononucleotide adenylyltransferase, a highly conserved enzyme which both catalyzes NAD+ synthesis in the last step of a salvage synthesis pathway that recycles nicotinamide (NAM) back to NAD+ by the chemical reaction of ATP + NMN to diphosphate + NAD+ but also can work to reverse this step in the NAD+/NADH biosynthetic pathway (1, 2, 7).

Nicotinamide adenine dinucleotide (NAD (NADP)) is a coenzyme found in all living cells and its derivatives are involved in many of the metabolic, both anabolic and catabolic, oxidation-reduction reactions including protein calcium signaling pathways, ADP-ribosylation, histone deacetylation, and many more. The mechanism of adenylytransfer by NMNATs begins with a nucleophilic attack on the 5'-phosphate of NMN at the -phosphate of ATP, which destabilizes the ester bond between the - and -phosphorous of ATP causing the NMN to be displaced at the -phosphate at which point the NAD+ and pyrophosphate are released from the enzyme (4).

While data on enzyme kinetics was found in reputable publications for all three human isoforms with regards to Km values for PPi , ATP, NAD+, and NMN, and Vmax values for NAD synthesis, NAD+ & NADH cleavage because there was only one source found that contained values not necessarily representing the overall kinetics with respects to all species the values were not mentioned in this article. More research will need to be completed to verify the values published are in fact an accurate representation of this enzyme's kinetics overall, not only Homo sapiens.

However, the values that were published do support all current research that of the three isoforms NMNAT-2 is the most highly conserved, is of the lowest concentration in the body and the most efficient of the three isoforms. NMNATs have gained recent attention due to the ability to delay neuronal degeneration induced by injury and it is part of the fusion protein in the Wallerian degeneration slow (wlds) mice that delays axonal degeneration after experimental transection, possibly due to mistargeting of the fusion protein to the cytoplasm (2, 7).

NMNATs reduction in activity by point mutation has been identified in the inherited form of retinal degeneration Leber congenital amaurosis and is currently the only monogenetic disease associated with NMNATs, and although only NMNAT1 was studied in this particular instance research seems to indicate NMNAT2 also plays an integral role in neuronal degeneration due to both injury and genetic conditions. It has been suggested that NMNATs are stress-responsive genes and are inducible by heat shock, hypoxia, and oxidative stress in the Drosophila model (2, 7). Potential use of NMNAT is for downstream targeting.

One study of the gene target p53 was consistent with providing data that shows the importance of NAD biosynthetic pathway and NMNATs importance in that process. In light of the multifaceted roles NAD+ play in mediating various aspects of cell physiology, cancer cells generally display aberrant or varying or altered NAD+ metabolism which allow them to proliferate and invade healthy tissues; one such example is known as the Warburg effect, which relates to cancer cells' dependence on cytoplasmic aerobic glycolysis, rather than the traditional mitochondrial oxidation-phosphorylation for production of energy (5).

The high rates of aerobic glycolysis disrupts the normal cellular NAD+ metabolism, which alters the NADH/NAD+ redox ratio, disrupting the cellular redox homeostasis which only further promotes cancer proliferation and progression (5). Because NMNAT-2 regulates cellular NAD+ level upon DNA damage, this study examined whether the induction of NMNAT-2 by p53 is indeed relevant to cellular NAD+ metabolism (5). The findings were that control cells had increased NAD+ levels by up to 50% upon DNA damage when compared with the non-treated cells, which is consistent with the idea that there is in fact, an elevated cellular demand for NAD+ under stressed conditions or upon DNA damage.

By knocking down both NMNAT-2 isoforms caused a drastic decrease of NAD+ level in comparison to non-treated cells, which also suggests there is a major role of NMNAT-2 proteins in replenishing the cellular NAD+ pool upon DNA damage. The data demonstrated that p53 facilitates cellular NAD+ biosynthesis through induction of NMNAT-2 to maintain a higher cellular NAD+ level under prolonged DNA damage condition and NMNAT-2 is involved in p53-mediated cell death where p53 is a major proapoptotic regulator of DNA damage-induced cell death (5). The degree that NMNAT-2 could play in future cancer treatment options is still unknown, but it is clear that because it plays an integral role in NAD metabolism, which occurs in all living organisms and cells, it will be an important enzyme in future biochemical technology research.

Another possible use of NMNAT is in regulation and regeneration of axons. While axon injury leads to several cell survival responses using an accessible Drosophila model, the regulation of injury responses was elucidated in a study which looked at axon regeneration and the role NMNAT plays in that model (6). The entire dendrite arbor is stabilized along with the rest of the cell upon axon injury and mitochondrial fission in dendrites was upregulated upon axon injury, so by reducing fission there was an increase in stabilization or neuroprotection (NP). In other words, axon injury seems to not only turn on NP but by activating mitochondrial fission it also dampens it (6). It has been determined that NMNAT is absolutely required for negative regulation of NP and NMNAT was required in the increased microtubule dynamics, which was previously associated with NP.

While NMNAT overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury the NP that occurs before axon regeneration, seems to be actively downregulated, so NMNAT overexpression reduces regeneration (6). Although further studies are required to see all the possible applications of NMNATs due to the vast nature of their expression, ability to regulate biosynthetic systems and pathways, and the implications of their importance in neurodegenerative disease and axon protection, it is clear that more is to be seen for this enzyme's use in the medical and scientific communities.