Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis.
Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.
Schweiger
M
M
Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
Hennig
K
K
Lerner
F
F
Niere
M
M
Hirsch-Kauffmann
M
M
Specht
T
T
Weise
C
C
Oei
S L
SL
Ziegler
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D009243:NAD / Q000096:biosynthesis*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D009612:Nicotinamide-Nucleotide Adenylyltransferase