Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

This review describes the enzymes involved in human pyridine nucleotide metabolism starting with a detailed consideration of their major kinetic, molecular and structural properties. The presentation encompasses all the reactions starting from the de novo pyridine ring formation and leading to nicotinamide adenine dinucleotide (NAD(+)) synthesis and utilization. The regulation of NAD(+) homeostasis with respect to the physiological role played by the enzymes both utilizing NAD(+) through the nonredox NAD(+)-dependent reactions and catalyzing the recycling of the common product, nicotinamide, is discussed. The salient features of other enzymes such as NAD(+) pyrophosphatase, nicotinamide mononucleotide 5'-nucleotidase, nicotinamide riboside kinase and nicotinamide riboside phosphorylase, described under 'miscellaneous', are likewise presented.

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Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines and other analogs using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7Å resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide’s amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed kcat/Km decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased Km for both NCA and AdoMet and modestly decreased kcat. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in Km(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

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