Evolutionary Analyses and Natural Selection of Betaine-Homocysteine S-Methyltransferase (BHMT) and BHMT2 Genes

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.VoRSUNY DownstateEpidemiology and BiostatisticsN/

Developmental and evolutionary analysis of betaine homocysteine methyltransferase genes

Methionine (Met) is an essential amino acid because mammals cannot synthesize homocysteine. Betaine-homocysteine methyltransferase (BHMT) and BHMT-2 methylate homocysteine to form methionine using betaine and S-methylmethionine, respectively. Betaine is produced de novo from choline and is also found in the diet, whereas S-methylmethionine is only made in plants and fungi and so must be obtained from the diet. These enzyme activities are only be detected in the liver of adult rodents, but in adult humans and pigs, these activities are found in both the liver and kidney cortex. Since both pigs and humans are omnivores and share the same pattern of BHMT and BHMT-2 expression, the pig represents an excellent model for studying the physiological roles of these enzymes in human biology. As a prelude to investigating the influence of diet and development on the expression of porcine BHMT and BHMT-2, their full-length cDNAs were cloned and sequenced, and their corresponding genes were characterized. The genes are adjacent to each other on the same chromosome, and to study the evolutionary relationship between them, all the available sequences from 37 species of deuterostomes were analyzed. Unlike BHMT, the BHMT-2 gene is not found in sea urchins, amphibians, reptiles and birds, indicating it was derived from BHMT following a gene duplication event in mammals. These findings imply that the BHMT-2 gene may offer an advantage to mammals in scavenging Met from the environment. As expected based on enzyme activity data from humans and pigs, BHMT and BHMT-2 mRNAs were observed to be highly expressed in liver and kidney cortex, whereas there is comparatively very little expression in other organs. The BHMT mRNA was higher in liver than kidney cortex (3:2 ratio), but the BHMT-2 mRNA was more abundant in kidney cortex than liver (3:1 ratio). The expression of BHMT mRNA was studied further. A total of ten different BHMT splice variants were observed in adult liver, kidney cortex, kidney medulla, lungs, heart, brain and fetal lungs. These included two variants that if translated would encode a truncated form of BHMT. BHMT mRNA expression was quantified during development at gestational week 30 (G30), G45, G90, and adult tissues. BHMT was low in G30 whole embryos, but was found to be easily quantifiable and progressively increased in the liver and kidney at and after G45. BHMT activity also progressively increased with age in both organs. The truncated transcripts represented ~10% of the total BHMT mRNA in the G30 fetus, the G45 liver and adult liver and kidney cortex. A computer-generated model of the truncated BHMT protein revealed a horseshoe fold structure of the protein, but the function of this putative protein is unknown. Using bisulfite sequencing, three CpG sites and the promoter region of the BHMT gene were identified that were more methylated in adult lungs compared to adult liver, suggesting that DNA methylation may be an important factor in the regulation of BHMT expression during development

Splicing variants of the porcine betaine–homocysteine S-methyltransferase gene: Implications for mammalian metabolism

Betaine–homocysteine S-methyltransferase (BHMT) activity is only detected in the liver of rodents, but in both the liver and kidney cortex of humans and pigs; therefore, the pig was chosen as a model to define the spatial and temporal expression of BHMT during development. During fetal development, a total of ten splice variants of bhmt were expressed at varying levels across a wide range of porcine tissues. Two variants contained an identical ORF that encoded a C-terminal truncated form of BHMT (tBHMT). The bhmt transcripts were expressed at significant levels in the liver and kidney from day 45 of gestation (G45) onward. The transcripts encoding tBHMT represented 5–13% of the total bhmt transcripts in G30 fetus, G45 liver, and adult liver and kidney cortex. The dominant structural feature of wild type BHMT is an (βα) 8 barrel, however, a modeled structure of tBHMT suggests that this protein would assume a horseshoe fold and lack methyltransferase activity. LowBHMT activity was detected in the G30 fetus, and slightly increased levels of activity were observed in the liver from G45 and G90 fetuses. The bhmt promoter contained three key CpG sites, and methylation of these sites was significantly higher in adult lung compared to adult liver. The data reported herein suggest that genomic DNA methylation and variation of the 5′ and 3′ UTRs of bhmt transcripts are key regulators for the level of BHMT transcription and translation