An assay for betaine-homocysteine methyltransferase activity based on the microbiological detection of methionine

Abstract

Betaine-homocysteine methyltransferase (BHMT: EC 2.1.1.5) catalyzes a methyl transfer from betaine to homocysteine (Hcy) forming dimethylglycine and methionine (Met), respectively. We have developed an assay for BHMT activity based on the microbiological detection of one of its products, Met. The microbiological assay uses Escherichia coli J5-3, a Met auxotroph. When cultured in minimal media, J5-3 growth increases linearly with Met supplementation up to 50 μM (2.24 μg/mL). Although not sensitive enough for kinetic analyses, this assay is adequate for measuring activity levels in crude or purified enzyme preparations using saturating substrate concentrations. When BHMT activity was measured in crude liver extracts, using both the radioactive and microbiological assays, the values obtained show a high degree of correlation ( r 2 > 0.93) over an eight-fold range of activities. We have used the microbiological assay to identify stachydrine as a naturally occurring methyl donor substrate for BHMT. The sensitivity of detecting BHMT activity by the microbiological assay for Met can be increased at least an order of magnitude by using dimethylacetothetin, a sulfonium analog of betaine, as the methyl donor substrate.