Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical characterization.
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and other pyridines. Human liver NNMT activity has a bimodal frequency distribution, an observation which raises the possibility that this enzyme activity might be regulated by a genetic polymorphism, a polymorphism that could have functional implications for individual differences in drug and xenobiotic toxicity. As a first step toward testing that hypothesis, we set out to clone and express a cDNA for human liver NNMT. Human liver NNMT was partially purified, photoaffinity-labeled, subjected to limited proteolysis, and partial amino acid sequence information was obtained. The polymerase chain reaction was then used to amplify a 550-nucleotide sequence with human liver cDNA as template and primers designed on the basis of the NNMT amino acid sequence. The 5'- and 3'-ends of a human liver NNMT cDNA were obtained by use of the rapid amplification of cDNA ends. The combined use of these approaches resulted in the isolation of a human liver NNMT cDNA that was 969 nucleotides in length, with a 792-nucleotide open reading frame that encoded a 264-amino acid protein with a calculated molecular mass of 29.6 kDa. The human liver NNMT cDNA was transcribed in vitro and translated with a reticulocyte lysate system to yield a protein with a molecular mass of approximately 29 kDa that comigrated during SDS-polyacrylamide gel electrophoresis with photoaffinity-labeled human liver NNMT. The NNMT cDNA was also subcloned into the eukaryotic expression vector p91023(B). COS-1 cells transfected with this construct expressed a high level of NNMT enzymatic activity, and the biochemical properties of this activity were similar to those of human liver NNMT. Human liver NNMT and transfected COS-1 cell NNMT had apparent Km values for the two cosubstrates for the reaction, nicotinamide and S-adenosyl-L-methionine, of 0.43 and 0.38 mM and of 1.8 and 2.2 microM, respectively. IC50 values for the inhibition of NNMT by N1-methylnicotinamide were 60 and 30 microns for human liver and COS-1 cell-expressed NNMT, respectively. Cloning of a cDNA for human liver NNMT will help make it possible to test the hypothesis that inheritance may play a role in the regulation of individual differences in human liver NNMT activity.
Aksoy
S
S
Department of Pharmacology, Mayo Medical School/Mayo Clinic, Rochester, Minnesota 55905.
Szumlanski
C L
CL
Weinshilboum
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008214:Lymphocytes / Q000201:enzymology; D008780:Methyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D050903:Nicotinamide N-Methyltransferase; D009928:Organ Specificity; D010802:Phylogeny; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D004247:DNA; D008780:Methyltransferases; C494406:NNMT protein, human; D050903:Nicotinamide N-Methyltransferase