Characterization of recombinant human nicotinamide mononucleotide adenylyltransferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential en zyme in all organisms, because it catalyzes a key step of NAD synthesis. Ho wever, little is known about the structure and regulation of this enzyme. I n this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzy me for an organism higher than yeast. The enzyme was purified from human pl acenta and internal peptide sequences determined, Analysis of human DNA seq uence data then permitted the cloning of a cDNA encoding this enzyme. Recom binant NMNAT exhibited catalytic properties similar to the originally purif ied enzyme, Human NMNAT (molecular weight 31932) consists of 279 amino acid s and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunof luorescence studies, NMNAT strongly inhibited recombinant human poly(ADP-ri bose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NM NAT appears to be a substrate of nuclear kinases and contains at least thre e potential phosphorylation sites. Endogenous and recombinant NMNAT were ph osphorylated in nuclear extracts in the presence of [gamma-P-32]ATP. We pro pose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation, (C) 2001 Federation of European Biochem ical Societies. Published hy Elsevier Science B.V. All rights reserved.