Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase.
Porcine liver betaine-homocysteine methyltransferase (BHMT; EC) was purified to homogeneity, and the Michaelis constants for betaine, dimethylacetothetin, and L-homocysteine are 23, 155, and 32 microM, respectively. The maximum rate of catalysis is 47-fold greater using dimethylacetothetin as a methyl donor compared with betaine. Partial amino acid sequence of porcine BHMT was obtained, and inosine-containing redundant oligonucleotide primers were used to amplify an 815-base pair sequence of the porcine cDNA by polymerase chain reaction (PCR). Nondegenerate oligonucleotide primers based on the porcine cDNA were synthesized and used to isolate a 463-base pair fragment of the human cDNA by PCR. The human PCR DNA product was then used to screen a cDNA library by plaque hybridization, and cDNAs encoding human BHMT were isolated. The primary structure of the human cDNA is reported here, and the open reading frame encodes a 406-residue protein of Mr 44,969. The deduced amino acid sequence of human BHMT shows limited homology to bacterial vitamin B12-dependent methionine synthases (EC). A plasmid containing the human BHMT cDNA fused in frame to the N terminus of beta-galactosidase was transformed into Escherichia coli, and transformants expressed BHMT activity, an activity that is absent from wild type E. coli.
Garrow
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Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D050940:Betaine-Homocysteine S-Methyltransferase; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013552:Swine
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase