Rate-limiting Steps in Folate Metabolism by Lactobacillus casei

Abstract

Oxidation of 5-methyltetrahydrofolate to 5,Io-methylenetetrahydrofolate was the ratelimiting step in 5-methyltetrahydrofolate metabolism by Lactobacillus casei. The limiting steps in the utilization of suboptimal levels of folate by L. casei were related to the ability of folates to function in purine and/or thymidylate biosynthesis. Folates with glutamate chains of up to at least seven residues were substrates for these biosynthetic enzymes, and comparisons of bacterial growth yields with transport rates for these folates indicated that the polyglutamates were more effective substrates in purine and thymidylate synthesis than the corresponding pteroylmonoglutamates. Lactobacillus casei contained low levels of a B,,-independent, pteroylpolyglutamate-specific methionine synthetase. Its methylenetetrahydrofolate reductase also functioned more effectively with pteroylpolyglutamate substrates. I N T R O D U C T I O NThe major route for 5-methyltetrahydrofolate (5-methyl-H4PteGlu) metabolism in Lactobacillus casei is oxidation with the incorporation of the one-carbon moiety of 5-methyl-H4PteGlu into thymidylate and purine derivatives (Shane & Stokstad, I 977).In this study, the growth requirement of L. casei for folates in media containing purines or thymine and the effects of products of one-carbon metabolism on 5-methyl-H4PteGlu metabolism were investigated to assess rate-limiting steps in folate metabolism. METHODSNomenclature. The abbreviations used are : PteGlu, pteroylglutamic acid, folk acid; PteGlu,, pteroylmono-to pteroyloligo-y-L-glutamic acid, n indicating the number of glutamic acid residues ; H,PteGlu,, 5,6,7,8-tetrahydropteroylmono-to 5,6,7,8-tetrahydropteroyloligo-y-~-glutamic acid. The symbols (l) and (d) are used to denote the natural and unnatural diastereoisomers of H,PteGlu,, respectively, due to the asymmetric centre at the C-6 position, and do not indicate optical activity. Materials. (I)-5-~Me-14C]Methyl-H4PteGlu, (specific activity, 47 mCi mmol-l), (l)-~o-~ormyl-~~C]formyl-H,PteGlu (sp. act., 47 mCi mmol-l), and PteGlu, (n = I, 3, 5 and 7) were synthesized and purified as described previously (Shane & Stokstad, 1976).[14C]Formate (sp. act., 47 mCi mmol-l) was obtained from Schwarz/Mann (Orangeburg, New Jersey, U.S.A.) and unlabelled purines and pyrimidines, nucleosides and amino acids from Sigma.Organism and growth conditions. Lactobacillus casei (~~~~7 4 6 9 ) was cultured as described previously (Tamura et al., 1972).Metabolism of (l)-5-[Me-14CJmethyl-H4PteGlu. Lactobacillus casei, harvested from growth media in late exponential phase, was washed with, and resuspended (0.2 mg dry wt ml-l) in 50 ~M-K~HPOJIOO msodium acetate, adjusted to pH 6 with H3P04, and containing I % (w/v) glucose and 5 m-mercaptoethanol.The bacteria were preincubated at 37 "C for z h before addition of (l)-5-[Me-14C]methyl-H,PteGlu (0.45 ,uM). After a further 15 min, the bacteria were filtered, resuspended in fresh buffer (0.4 mg dry wt ml-l) and reincubated at 37 "C for 2 h. Intracellular labelled metabolites were extr...

Folate status assessment history: implications for measurement of biomarkers in NHANES

“…A major advantage of assays with L. casei is that L. casei responds equally well to all folate monoglutamate forms ( Table 1 ) (8). Although the affinity for the folate transporter varies with different monoglutamates, growth promotion is essentially identical because intracellular one-carbon metabolism, rather than transport, limits growth on monoglutamates (13, 14). …”

“…L. casei exhibits a growth response not only to folate but also to products of one-carbon metabolism, and grows in the complete absence of folate if the medium contains thymidine, purines, methionine, serine, glycine, and pantothenate (14). Human plasma contains significant concentrations of all of these compounds, with the exception of thymidine.…”

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis

“…1.1 mM), all derived from a pancreatin digest of casein, and uracil (45 ,uM), but lacked purines and thymine. Under these conditions, folate was required primarily for the biosynthesis of purines and thymidylate (32,33).…”

The threshold growth response ofLactobacillus caseito 5-methyl-tetrahydrofolic acid: implications for folate assays

“…The metabolism of 5-methyl-tetrahydrofolic acid-monoglutamate by L. casei can only proceed through its direct oxidation by 5, 10-methylene-tetrahydrofolate reductase (FADH,) (EC 1.1.99.15) to 5, 10-methylene-tetrahydrofolic acid and its subsequent utilization in thymidylate and purine synthesis (Shane & Stokstad, 1977 a). However, the equilibrium of 5, 10-methylene-tetrahydrofolic acid reductase can be strongly against this reaction (Shane & Stokstad, 1977b) and, as this is the only step that 5-methyl-tetrahydrofolic acid metabolism does not share with folic acid metabolism, it would appear that when L. casei is in a medium at pH 6.8 with low substrate concentrations, the amount of transported folate is insufficient to activate this enzyme (thus trapping folate unmetabolized and causing a threshold effect on growth) and that oxidation only occurs when the intracellular concentration is sufficient to overcome the enzyme equilibrium.…”

“…casei requires folate primarily for the generation of purines and thymidylate, their synthesis being thought to be the most rate-limiting steps governing growth (Shane & Stokstad, 1977b). Folic acid uptake by L. casei is independent of conversion to other folate forms (Cooper, 1970), and enters the folate metabolic cycle via reduction to tetrahydrofolic acid which can be subsequently metabolized to 5, 10-methylene-tetrahydrofolic acid, the one-carbon moiety of which is rapidly incorporated into thymidylate and purines.…”