Hepatic Activity and Transcription of Betaine-Homocysteine Methyltransferase, Methionine Synthase, and Cystathionine Synthase in Periparturient Dairy Cows Are Altered to Different Extents by Supply of Methionine and Choline.

BACKGROUND: Compared with choline, Met enhances milk yield and feed intake, and elicits a better immuno-metabolic status in periparturient cows. It is unknown whether hepatic activity and transcription of betaine-homocysteine methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine ß-synthase (CBS) are responsive to Met and choline supply. OBJECTIVE: This study sought to characterize hepatic BHMT, MTR, and CBS transcription and activity in response to Met and choline supplementation. METHODS: Forty multiparous cows were used in a 2 × 2 factorial design from -21 d through 30 d around parturition to assess effects of dietary rumen-protected Met (0% or 0.08% dry matter basis) or rumen-protected choline (0 or 60 g · cow-1 · d-1). Liver tissue obtained on days -10, 7, 20, and 30 was used for analyses. RESULTS: Met-supplemented cows had greater methionine adenosyltransferase 1A (MAT1A) (0.38 compared with 0.27; SEM = 0.05; P = 0.02) and phosphatidylethanolamine methyltransferase (PEMT) (0.74 compared with 0.58; SEM = 0.08; P = 0.05) expression. Greater S-adenosylhomocysteine hydrolase (SAHH) (0.93 compared with 0.74; SEM = 0.05; P = 0.01) and CBS (1.16 compared with 1.02; SEM = 0.07; P = 0.04), as well as lower MTR activity (23.4 compared with 29.7 nmol product · h-1 · mg protein-1; SEM = 2.9; P = 0.04), also were detected in Met- but not choline-supplemented cows. Although BHMT and MTR expression and BHMT enzyme activity did not change (P > 0.05), MTR enzyme activity was lower in choline-supplemented cows (23.5 compared with 29.6 nmol product · h-1 · mg protein-1; SEM = 2.9; P = 0.05). CONCLUSIONS: These findings indicate that greater synthesis of phosphatidylcholine and antioxidants contribute to the better performance and immuno-metabolic status in Met-supplemented cows. Failure to generate a comparable amount of endogenous Met from choline could be one reason that choline-fed cows fail to achieve comparable performance and health benefits during the periparturient period.

Specific potassium ion interactions facilitate homocysteine binding to betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S-adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K(M) for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K(M) of the enzyme for homocysteine, but it does not affect the apparent K(M) for betaine or the apparent k(cat) for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26-Gly27-Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site-specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme.

Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.

Deletion of betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinomas.

Betaine-homocysteine S-methyltransferase (BHMT) uses betaine to catalyze the conversion of homocysteine (Hcy) to methionine. There are common genetic polymorphisms in the BHMT gene in humans that can alter its enzymatic activity. We generated the first Bhmt(-/-) mouse to model the functional effects of mutations that result in reduced BHMT activity. Deletion of Bhmt resulted in a 6-fold increase (p < 0.01) in hepatic and an 8-fold increase (p < 0.01) in plasma total Hcy concentrations. Deletion of Bhmt resulted in a 43% reduction in hepatic S-adenosylmethionine (AdoMet) (p < 0.01) and a 3-fold increase in hepatic S-adenosylhomocysteine (AdoHcy) (p < 0.01) concentrations, resulting in a 75% reduction in methylation potential (AdoMet:AdoHcy) (p < 0.01). Bhmt(-/-) mice accumulated betaine in most tissues, including a 21-fold increase in the liver concentration compared with wild type (WT) (p < 0.01). These mice had lower concentrations of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin in several tissues. At 5 weeks of age, Bhmt(-/-) mice had 36% lower total hepatic phospholipid concentrations and a 6-fold increase in hepatic triacyglycerol concentrations compared with WT (p < 0.01), which was due to a decrease in the secretion of very low density lipoproteins. At 1 year of age, 64% of Bhmt(-/-) mice had visible hepatic tumors. Histopathological analysis revealed that Bhmt(-/-) mice developed hepatocellular carcinoma or carcinoma precursors. These results indicate that BHMT has an important role in Hcy, choline, and one-carbon homeostasis. A lack of Bhmt also affects susceptibility to fatty liver and hepatocellular carcinoma. We suggest that functional polymorphisms in BHMT that significantly reduce activity may have similar effects in humans.

Liver betaine-homocysteine S-methyltransferase activity undergoes a redox switch at the active site zinc.

Using a redox-inert methyl acceptor, we show that betaine-homocysteine S-methyltransferase (BHMT) requires a thiol reducing agent for activity. Short-term exposure of BHMT to reducing agent-free buffer inactivates the enzyme without causing any loss of its catalytic zinc. Activity can be completely restored by the re-addition of a thiol reducing agent. The catalytic zinc of BHMT is bound by three thiolates and one hydroxyl group. Thiol modification experiments indicate that a disulfide bond is formed between two of the three zinc-binding ligands when BHMT is inactive in a reducing agent-free buffer, and that this disulfide can be readily reduced with the concomitant restoration of activity by re-establishing reducing conditions. Long-term exposure of BHMT to reducing agent-free buffer results in the slow, irreversible loss of its catalytic Zn and a corresponding loss of activity. Experiments using the glutamate-cysteine ligase modifier subunit knockout mice Gclm(-/-), which are severely impaired in glutathione synthesis, show that BHMT activity is reduced about 75% in Gclm(-/-) compared to Gclm(+/+) mice.

Type I diabetes leads to tissue-specific DNA hypomethylation in male rats.

Numerous perturbations of methyl group and homocysteine metabolism have been documented as an outcome of diabetes. It has also been observed that there is a transition from hypo- to hyperhomocysteinemia in diabetes, often concurrent with the development of nephropathy. The objective of this study was to characterize the temporal changes in methyl group and homocysteine metabolism in the liver and kidney and to determine the impact these alterations have on DNA methylation in type 1 diabetic rats. Male Sprague-Dawley rats were injected with streptozotocin (60 mg/kg body weight) to induce diabetes and samples were collected at 2, 4, and 8 wk. At 8 wk, hepatic and renal betaine-homocysteine S-methyltransferase activities were greater in diabetic rats, whereas methionine synthase activity was lower in diabetic rat liver and kidney did not differ. Cystathionine beta-synthase abundance was greater in the liver but less in the kidney of diabetic rats. Both hepatic and renal glycine N-methyltransferase (GNMT) activity and abundance were greater in diabetic rats; however, changes in renal activity and/or abundance were present only at 2 and 4 wk, whereas hepatic GNMT was induced at all time points. Most importantly, we have shown that genomic DNA was hypomethylated in the liver, but not the kidney, in diabetic rats. These results suggest that diabetes-induced perturbations of methyl group and homocysteine metabolism lead to functional methyl deficiency, resulting in the hypomethylation of DNA in a tissue-specific fashion.

S-alkylated homocysteine derivatives: new inhibitors of human betaine-homocysteine S-methyltransferase.

A series of S-alkylated derivatives of homocysteine were synthesized and characterized as inhibitors of human recombinant betaine-homocysteine S-methyltransferase (BHMT). Some of these compounds inhibit BHMT with IC50 values in the nanomolar range. BHMT is very sensitive to the structure of substituents on the sulfur atom of homocysteine. The S-carboxybutyl and S-carboxypentyl derivatives make the most potent inhibitors, and an additional sulfur atom in the alkyl chain is well tolerated. The respective (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic, (R,S)-6-(3-amino-3-carboxy-propylsulfanyl)-hexanoic, and (R,S)-2-amino-4-(2-carboxymethylsulfanyl-ethylsulfanyl)-butyric acids are very potent inhibitors and are the strongest ever reported. We determined that (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid displays competitive inhibition with respect to betaine binding with a Kappi of 12 nM. Some of these compounds are currently being tested in mice to study the influence of BHMT on the metabolism of sulfur amino acids in vivo.

Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro.

Betaine-homocysteine S-methyltransferase (BHMT) transfers a methyl group from betaine to Hcy to form DMG (dimethylglycine) and Met. The reaction is ordered Bi Bi; Hcy is the first substrate to bind and Met is the last product off. Using intrinsic tryptophan fluorescence [Castro, Gratson, Evans, Jiracek, Collinsova, Ludwig and Garrow (2004) Biochemistry 43, 5341-5351], it was shown that BHMT exists in three steady-state conformations: enzyme alone, enzyme plus occupancy at the first substrate-binding site (Hcy or Met), or enzyme plus occupancy at both substrate-binding sites (Hcy plus betaine, or Hcy plus DMG). Betaine or DMG alone do not bind to the enzyme, indicating that the conformational change associated with Hcy binding creates the betaine-binding site. CBHcy [S-(d-carboxybutyl)-D,L-homocysteine] is a bisubstrate analogue that causes BHMT to adopt the same conformation as the ternary complexes. We report that BHMT is susceptible to conformation-dependent oxidative inactivation. Two oxidants, MMTS (methyl methanethiosulphonate) and hydrogen peroxide (H2O2), cause a loss of the enzyme's catalytic Zn (Zn2+ ion) and a correlative loss of activity. Addition of 2-mercaptoethanol and exogenous Zn after MMTS treatment restores activity, but oxidation due to H2O2 is irreversible. CD and glutaraldehyde cross-linking indicate that H2O2 treatment causes small perturbations in secondary structure but no change in quaternary structure. Oxidation is attenuated when both binding sites are occupied by CBHcy, but Met alone has no effect. Partial digestion of ligand-free BHMT with trypsin produces two large peptides, excising a seven-residue peptide within loop L2. CBHcy but not Met binding slows down proteolysis by trypsin. These findings suggest that L2 is involved in the conformational change associated with occupancy at the betaine-binding site and that this conformational change and/or occupancy at both ligand-binding sites protect the enzyme from oxidative inactivation.

Betaine-homocysteine methyltransferase: zinc in a distorted barrel.

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.

Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine: homocysteine S-methyltransferase.

A new method to find novel protein targets for ligands of interest is proposed. The principle of this approach is based on affinity chromatography and combinatorial chemistry. The proteins within a crude rat liver homogenate were allowed to interact with a combinatorial library of phosphinic pseudopeptides immobilized on affinity columns. Betaine: homocysteine S-methyltransferase (BHMT) was one of the proteins that was retained and subsequently eluted from these supports. The phosphinic pseudopeptides, which served as immobilized ligands for the isolation of rat BHMT, were then tested for their ability to inhibit human recombinant BHMT in solution. The most potent inhibitor also behaved as a selective ligand for the affinity purification of BHMT from a complex media. Further optimization uncovered Val-Phe-psi[PO(2-)-CH(2)]-Leu-His-NH(2) as a potent BHMT inhibitor that has an IC(50) of about 1 microM.

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.

Investigations of a common genetic variant in betaine-homocysteine methyltransferase (BHMT) in coronary artery disease.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, can be caused by genetic mutations in enzymes of homocysteine metabolism. Homocysteine remethylation to methionine is catalyzed by folate-dependent methionine synthase, or by betaine-homocysteine methyltransferase (BHMT), which utilizes betaine as the methyl donor. Since genetic variants in folate-dependent remethylation have been reported to increase risk for cardiovascular disease and other common disorders, we screened BHMT for sequence changes that might alter risk for coronary artery disease (CAD). A variant in exon 6-R239Q-was identified. The frequency of this change was examined in 504 individuals who had undergone coronary angiography and were stratified into controls (those with no or mild disease) and cases (those with significant [>50% reduction in luminal diameter stenosis] 1-, 2-, 3-vessel disease). Although this variant did not affect plasma homocysteine, the QQ genotype was present in higher frequency in those with no or mild disease, compared with those with significant disease (11 vs. 6%), suggesting that it may decrease risk of CAD; a statistically-significant decrease was seen in the older subjects (13 vs. 7%). Multivariate analysis for the entire group revealed an odds ratio of 0.48 (95% CI: 0.21-1.06) for the QQ genotype; this association was similar in the younger (OR=0.36; 95% CI: 0.09-1.41) and older subjects (OR=0.42; 95% CI: 0.15-1.18). Our study suggests that the Q allele of the R239Q mutation may decrease the risk of CAD and that this variant warrants additional investigation of its relationship with the development of CAD as well as other homocysteine-dependent disorders.

Effects of betaine in a murine model of mild cystathionine-beta-synthase deficiency.

Cystathionine-beta-synthase (CBS) is required for transsulfuration of homocysteine, an amino acid implicated in vascular disease. We studied homocysteine metabolism in mice with mild hyperhomocysteinemia due to a heterozygous disruption of the Cbs gene. Mice were fed diets supplemented with betaine or dimethylsulfonioacetate (DMSA); betaine and DMSA provide methyl groups for an alternate pathway of homocysteine metabolism, remethylation by betaine:homocysteine methyltransferase (BHMT). On control diets, heterozygous mice had 50% higher plasma homocysteine than did wild-type mice. Betaine and DMSA had similar effects in both genotype groups: liver betaine increased dramatically, while plasma homocysteine decreased by 40% to 50%. With increasing betaine supplementation, homocysteine decreased by 75%. Plasma homocysteine and BHMT activity both showed a strong negative correlation with liver betaine. Homocysteinemia in mice is sensitive to a disruption of Cbs and to methyl donor intake. Because betaine leads to a greater flux through BHMT and lowers homocysteine, betaine supplementation may be beneficial in mild hyperhomocysteinemia.

Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used for determination of dissociation constant of phosphinate group in phosphinic pseudopeptides, i.e. peptides where one peptide bond is substituted by phosphinic acid moiety -PO2--CH2-. The dissociation constants were determined for a set of newly synthesized pseudopeptides derived from a structure N-Ac-Val-Ala(psi)(PO2--CH2)Leu-His-NH2 by nonlinear regression of experimentally measured pH dependence of their effective electrophoretic mobilities. CZE experiments were carried out in Tris-phosphate background electrolytes in the pH range 1.4-3.2. The pseudopeptides were synthesized as a mixture of four diastereomers, the separation of which was achieved in most cases. Moreover, differences of the effective mobilities of the pseudopeptide diastereomers enabled simultaneous determination of the dissociation constant of their phosphinate group without necessity of previous isolation of individual isomers.

The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) is an important zinc-dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. In the liver, BHMT performs to half of the homocysteine remethylation. In this study, we systematically investigated the tolerance of the enzyme for modifications at the "homocysteine" part of the previously reported potent inhibitor (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid (1). In the new compounds, which are S-alkylated homocysteine derivatives, we replaced the carboxylic group in the "homocysteine" part of inhibitor 1 with different isosteric moieties (tetrazole and oxadiazolone); we suppressed the carboxylic negative charge by amidations; we enhanced acidity by replacing the carboxylate with phosphonic or phosphinic acids; and we introduced pyrrolidine steric constraints. Some of these compounds display high affinity toward human BHMT and may be useful for further pharmacological studies of this enzyme. Although none of the new compounds were more potent inhibitors than the reference inhibitor 1, this study helped to completely define the structural requirements of the active site of BHMT and revealed the remarkable selectivity of the enzyme for homocysteine.

Double-headed sulfur-linked amino acids as first inhibitors for betaine-homocysteine S-methyltransferase 2.

Betaine-homocysteine S-methyltransferase 2 (BHMT-2) catalyzes the transfer of a methyl group from S-methylmethionine to l-homocysteine, yielding two molecules of l-methionine. It is one of three homocysteine methyltransferases in mammals, but its overall contribution to homocysteine remethylation and sulfur amino acid homeostasis is not known. Moreover, recombinant BHMT-2 is highly unstable, which has slowed research on its structural and catalytic properties. In this study, we have prepared the first series of BHMT-2 inhibitors to be described, and we have tested them with human recombinant BHMT-2 that has been stabilized by copurification with human recombinant BHMT. Among the compounds synthesized, (2S,8RS,11RS)-5-thia-2,11-diamino-8-methyldodecanedioic acid (11) was the most potent (K(i)(app) ∼77 nM) and selective inhibitor of BHMT-2. Compound 11 only weakly inhibited human BHMT (IC(50) about 77 µM). This compound (11) may be useful in future in vivo studies to probe the physiological significance of BHMT-2 in sulfur amino acid metabolism.

Molecular characterization and analysis of the porcine betaine homocysteine methyltransferase and betaine homocysteine methyltransferase-2 genes.

Betaine homocysteine methyltransferase (BHMT) and BHMT-2 enzymes methylate homocysteine to form methionine using betaine and S-methylmethionine, respectively. These activities are observed only in the liver of adult rodents, but in adult humans and pigs these activities are detected in both the liver and kidney, indicating the pig is a more appropriate model for studying the biochemical and physiological roles of these enzymes in human biology. Porcine BHMT and BHMT-2 cDNAs were cloned and sequenced, and their 5' and 3' UTR were amplified using RLM-RACE. The BHMT transcript had significantly longer 5' and 3' UTRs than BHMT-2. The pig BHMT and BHMT-2 genes span approximately 26 and 16kb, respectively, and both genes have 8 exons. The deduced amino acid sequences of BHMT and BHMT-2 contain 407 and 363 amino acids, respectively, and shared 78% amino acid identity. No promoter element (TATA or CAAT box) was observed for either BHMT or BHMT-2, although a CpG island surrounding the promoter and transcriptional start site was observed in both genes implying that methylation could regulate their expression. Using qPCR, it was determined that BHMT and BHMT-2 transcripts are very abundant in liver and kidney cortex, whereas the expression is significantly less in other tissues. These findings confirm that the expression pattern of BHMT and BHMT-2 genes in pigs is similar to humans, supporting the use of the pig as an animal model to study the genetics and regulation of BHMT and BHMT-2 expression.

Inhibition of betaine-homocysteine S-methyltransferase in rats causes hyperhomocysteinemia and reduces liver cystathionine ß-synthase activity and methylation capacity.

Methylation of homocysteine (Hcy) by betaine-Hcy S-methyltransferase (BHMT) produces methionine, which is required for S-adenosylmethionine (SAM) synthesis. We have recently shown that short-term dietary intake of S-(Δ-carboxybutyl)-dl-Hcy (D,L-CBHcy), a potent and specific inhibitor of BHMT, significantly decreases liver BHMT activity and SAM concentrations but does not have an adverse affect on liver histopathology, plasma markers of liver damage, or DNA methylation in rats. The present study was designed to investigate the hypothesis that BHMT is required to maintain normal liver and plasma amino acid and glutathione profiles, and liver SAM and lipid accumulation. Rats were fed an adequate (4.5 g/kg methionine and 3.7 g/kg cystine), cysteine-devoid (4.5 g/kg methionine and 0 g/kg cystine), or methionine-deficient (1.5 g/kg methionine and 3.7 g/kg cystine) diet either with or without L-CBHcy for 3 or 14 days. All rats fed L-CBHcy had increased total plasma Hcy (2- to 5-fold) and reduced liver BHMT activity (>90%) and SAM concentrations (>40%). S-(Δ-carboxybutyl)-l-Hcy treatment slightly reduced liver glutathione levels in rats fed the adequate or cysteine-devoid diet for 14 days. Rats fed the methionine-deficient diet with L-CBHcy developed fatty liver. Liver cystathionine ß-synthase activity was reduced in all L-CBHcy-treated animals, and the effect was exacerbated as time on the L-CBHcy diet increased. Our data indicate that BHMT activity is required to maintain adequate levels of liver SAM and low levels of total plasma Hcy and might be critical for liver glutathione and triglyceride homeostasis under some dietary conditions.

Dietary intake of S-(alpha-carboxybutyl)-DL-homocysteine induces hyperhomocysteinemia in rats.

Betaine homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine (Hcy), forming dimethylglycine and methionine. We previously showed that inhibiting BHMT in mice by intraperitoneal injection of S-(alpha-carboxybutyl)-DL-homocysteine (CBHcy) results in hyperhomocysteinemia. In the present study, CBHcy was fed to rats to determine whether it could be absorbed and cause hyperhomocysteinemia as observed in the intraperitoneal administration of the compound in mice. We hypothesized that dietary administered CBHcy will be absorbed and will result in the inhibition of BHMT and cause hyperhomocysteinemia. Rats were meal-fed every 8 hours an L-amino acid-defined diet either containing or devoid of CBHcy (5 mg per meal) for 3 days. The treatment decreased liver BHMT activity by 90% and had no effect on methionine synthase, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase, and CTP:phosphocholine cytidylyltransferase activities. In contrast, cystathionine beta-synthase activity and immunodetectable protein decreased (56% and 26%, respectively) and glycine N-methyltransferase activity increased (52%) in CBHcy-treated rats. Liver S-adenosylmethionine levels decreased by 25% in CBHcy-treated rats, and S-adenosylhomocysteine levels did not change. Furthermore, plasma choline decreased (22%) and plasma betaine increased (15-fold) in CBHcy-treated rats. The treatment had no effect on global DNA and CpG island methylation, liver histology, and plasma markers of liver damage. We conclude that CBHcy-mediated BHMT inhibition causes an elevation in total plasma Hcy that is not normalized by the folate-dependent conversion of Hcy to methionine. Furthermore, metabolic changes caused by BHMT inhibition affect cystathionine beta-synthase and glycine N-methyltransferase activities, which further deteriorate plasma Hcy levels.

Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase.

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(CH(3)), or N(CH(3))(2) groups separated from the homocysteine sulfur atom by a methylene, ethylene, or a propylene spacer. Only the inhibitor with the N(CH(3)) 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.