Nicotinamide mononucleotide adenylyltransferase. Molecular and enzymic properties of the homogeneous enzyme from bakers' yeast

Abstract

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4.

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

“…3B). This pattern of inhibition indicates that Pof1 may exhibit an ordered sequential mechanism, which was also observed with human NMNAT1 (27,28). Interestingly, Pof1 displayed an alkaline optimum pH (pH 10).…”

“…Precedents exist for efficient enzymes that exhibit high K m for their substrates. For example, Nma1 has high K m for NaMN (ϳ5 mM) but is a key enzyme in the de novo and NA salvage pathways for NAD ϩ synthesis (27), and deletion of Nma1 significantly decreases NAD ϩ levels (12). Our study also suggested that Pof1 activity is specific for NMN unlike Nma1 and Nma2, which exhibit dual substrate specificity toward NMN and NaMN (Fig.…”

Identification, Characterization, and Crystal Structure ofBacillus subtilis Nicotinic Acid Mononucleotide Adenylyltransferase

“…Two of the enzymes, those from M. jannaschii (16) and M. thermoautotrophicum (17), have been shown to crystallize as homohexamers, and a third from Synechocystits was identified as a homohexamer by size exclusion chromatography (33). The remaining five, E. coli nadR (14), and the NMN ATs from Sulfolobus solfataricus (11), Saccharomyces cerevisiae (7,8), bull testes (5), and human (9, 10) appear to be trimers or tetramers by size exclusion chromatography. If the two crystal structures presented here reflect the physiological oligomeric state of NaMN AT, then the functional dimer, which is conserved in the independent crystal environments of the apo and NaAD-bound form, is proposed to be the dimer observed in solution.…”

Enzymology of Nad + Synthesis

“…Among eukaryotes, NMN adenylyltransferase has been partially purified from pig liver nuclei (Ferro and Kuehl, 1975), chicken erythrocytes (Cantarow and Stollar, 1977a), and skipjack (Kono et al, 1978). The first homogeneous enzymatic preparation was obtained from the yeast Succhuromyces cerevisiae (Natalini et al, 1986). Later the enzyme was purified to homogeneity and extensively characterized in its molecular and kinetic properties from bull testis (Balducci et al, 1995) and human placenta .…”