Purification, Kinetic Properties, and cDNA Cloning of Mammalian Betaine-Homocysteine Methyltransferase

Abstract

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 µM, 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 <i>M</i>_r 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 β-galactosidase was transformed into <i>Escherichia coli</i>, and transformants expressed BHMT activity, an activity that is absent from wild type <i>E. coli</i>.