PURIFICATION, KINETIC-PROPERTIES, AND CDNA CLONING OF MAMMALIAN BETAINE-HOMOCYSTEINE METHYLTRANSFERASE

Porcine liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5 ) was purified to homogeneity, and the Michaelis constants for betaine , dimethylacetothetin, and L-homocysteine are 23, 155, and 32 mu M, re spectively. The maximum rate of catalysis is 47-fold greater using dim ethylacetothetin as a methyl donor compared with betaine. Partial amin o acid sequence of porcine BHMT was obtained, and inosine containing r edundant oligonucleotide primers were used to amplify an 815-base pair sequence of the porcine cDNA by polymerase chain reaction (PCR). Nond egenerate oligonucleotide primers based on the porcine cDNA were synth esized 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 libra ry by plaque hybridization, and cDNAs encoding human BHMT were isolate d. The primary structure of the human cDNA is reported here, and the o pen reading frame encodes a 406-residue protein of M(r) 44,969. The de duced amino acid sequence of human BHMT shows limited homology to bact erial vitamin B-12-dependent methionine synthases (EC 2.1.1.13). A pla smid containing the human BHMT cDNA fused in frame to the N terminus o f beta-galactosidase was transformed into Escherichia coli, and transf ormants expressed BHMT activity, an activity that is absent from wild type E. coli.