Folic acid and the methylation of homocysteine by Bacillus subtilis

Abstract

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C(1) transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B(12). 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.

Identification of poly-gamma-glutamyl chain lengths in folates of Bacillus subtilis

“…The two large peaks observed in elution patterns from G-25 columns must both be derivatives of pteglu3. It is likely that one of the peaks is the N5-formyl derivative, since that compound has previously been identified as a major folate compound in B. subtilis (7). The identity of the other derivative is unknown.…”

“…Bacillus subtilis has been reported to contain pteroyltriglutamate (pteglu3) (6, 7) and another unidentified folate (7). Recently, a technique has been developed which allows positive identification of the number of glutamate residues contained in each of the folate types of an organism (2).…”

Composite genome sequence of Bacillus clausii, a probiotic commercially available as Enterogermina®, and insights into its probiotic properties

“…Therefore, it is expected that these strains are not auxotrophic for folates or DHP but can produce folate in the presence of PABA supplementation.The presence/absence of the components of the folate biosynthesis pathway is reported based on KEGG pathway analysis. Previous studies have revealed that B. subtilis genome harbor all the pathways components and have been engineered for folate production[63,65,66].…”

Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

“…The rate-limiting step in the utilization of pteroylpolyglutamates by this organism is transport into the cell, while intracellular metabolism, and not transport, is rate-limiting with pteroylmonoglutamates. In vitro studies have also shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (Burton & Metzenberg, 1975;Cheng, Shane & Stokstad, 1975;Coward et al, 1974 Coward et al, , 1975Curthoys & Rabinowitz, 1972;Kisliuk, Gaumont & Baugh, 1974;Powers & Snell, 1976;Salem, Pattison & Foster, 1972;Whitfield, Steers & Weissbach, 1970).…”

“…The rate-limiting step in the utilization of pteroylpolyglutamates by this organism is transport into the cell, while intracellular metabolism, and not transport, is rate-limiting with pteroylmonoglutamates. In vitro studies have also shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (Burton & Metzenberg, 1975;Cheng, Shane & Stokstad, 1975;Coward et al, 1974 Coward et al, , 1975Curthoys & Rabinowitz, 1972;Kisliuk, Gaumont & Baugh, 1974;Powers & Snell, 1976;Salem, Pattison & Foster, 1972;Whitfield, Steers & Weissbach, 1970).To investigate which step in pteroylmonoglutamate metabolism might be rate-limiting, the metabolism of 5-[Me-14C]methyl-H,PteGlu and 5-methyl-H,[3H]PteGlu (for nomenclature, see Methods) were compared in order to assess the turnover of intracellular folate via various metabolic pathways. Although two mechanisms have been described for 5-methyl-H4PteGlu formation in bacteria involving the reduction of 5, I o-methylene-H,PteGlu or the direct transfer of a methyl group from trimethylsulphonium to H,PteGlu (Wagner et al, 1967), the only known route for the metabolism of methyltetrahydrofolates is via methionine synthesis (Taylor & Weissbach, 1973) but attempts to detect methionine synthetase activity in L. casei have proved unsuccessful (Galivan, 1971 ;Kisliuk, 1971).…”