A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria.
BHMT transfers a methyl group from betaine (Bet) to homocysteine to form Met and dimethylglycine. Bet is obtained from the diet or by the endogenous oxidation of choline by CHDH. BHMT is expressed in rat liver and is regulated by dietary Met and choline. BHMT is also found in rat kidney, albeit in substantially lower amounts, but it is not known whether it is regulated by dietary Met or choline. Whether diet has a role in the regulation of CHDH expression has not been investigated. Rats (~50g) were fed (9 days) in 3 x 2 factorial design (n = 8) a purified diet varying in Met (0.125, 0.3 or 0.8%) and choline (0 or 0.625%). Liver and kidney BHMT and CHCH expression were assessed using enzymatic, Western blot and real-time PCR analyses. Livers were also fixed in 10% buffered formalin for histological analysis. Animals fed low Met diets had significantly lower feed efficiencies. A degree of fatty liver was observed in all rats fed a choline-devoid diet, indicating that excess Met cannot compensate for low dietary choline. Compared to rats fed the 0.3% Met and 0.625% choline diet, liver BHMT activity was 113% higher in animals fed the 0.125% Met containing 0.625% choline and was also reflected in increases in mRNA content (2.7-fold) and immunodetectable protein. Excess dietary Met with or without choline increased liver BHMT activity 19.7% to 33.3%. Kidney BHMT was refractory to dietary treatment. Liver CHDH immunodetectable protein and mRNA content were not affected by dietary Met and choline availability. This work was supported by NIDDK (DK52501).
Betaine-homocysteine S-methyltransferase (BHMT) and BHMT2 convert homocysteine to methionine using betaine and S-methylmethionine, respectively, as methyl donor substrates. Increased levels of homocysteine in blood are associated with cardiovascular disease. Given their role in human health and nutrition, we identified BHMT and BHMT2 genes and proteins from 38 species of deuterostomes including human and non-human primates. We aligned the genes to look for signatures of selection, to infer evolutionary rates and events across lineages, and to identify the evolutionary timing of a gene duplication event that gave rise to two genes, BHMT and BHMT2. We found that BHMT was present in the genomes of the sea urchin, amphibians, reptiles, birds and mammals; BHMT2 was present only across mammals. BHMT and BHMT2 were present in tandem in the genomes of all monotreme, marsupial and placental species examined. Evolutionary rates were accelerated for BHMT2 relative to BHMT. Selective pressure varied across lineages, with the highest dN/dS ratios for BHMT and BHMT2 occurring immediately following the gene duplication event, as determined using GA Branch analysis. Nine codons were found to display signatures suggestive of positive selection; these contribute to the enzymatic or oligomerization domains, suggesting involvement in enzyme function. Gene duplication likely occurred after the divergence of mammals from other vertebrates but prior to the divergence of extant mammalian subclasses, followed by two deletions in BHMT2 that affect oligomerization and methyl donor specificity. The faster evolutionary rate of BHMT2 overall suggests that selective constraints were reduced relative to BHMT. The dN/dS ratios in both BHMT and BHMT2 was highest following the gene duplication, suggesting that purifying selection played a lesser role as the two paralogs diverged in function.
Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to l-homocysteine, yielding dimethylglycine and l-methionine. In this study, we prepared a new series of BHMT inhibitors. The inhibitors were designed to mimic the hypothetical transition state of BHMT substrates and consisted of analogues with NH, N(CH3), or N(CH3)2 groups separated from the homocysteine sulfur atom by a methylene, ethylene, or a propylene spacer. Only the inhibitor with the N(CH3) moiety and ethylene spacer gave moderate inhibition. This result led us to prepare two inhibitors lacking a nitrogen atom in the S-linked alkyl chain: (RS,RS)-5-(3-amino-3-carboxypropylthio)-3-methylpentanoic acid and (RS)-5-(3-amino-3-carboxypropylthio)-3,3-dimethylpentanoic acid. Both of these compounds were highly potent inhibitors of BHMT. The finding that BHMT does not tolerate a true betaine mimic within these inhibitors, especially the nitrogen atom, is surprising and evokes questions about putative conformational changes of BHMT upon the binding of the substrates/products and inhibitors.
