aminomethyltransferase and 5-6-7-8-tetrahydrofolic-acid

Expression of yeast genes involved in one-carbon metabolism is controlled by glycine, by L-methionine, and by nitrogen sources. Here we report a novel control element containing a core CTTCTT motif mediating the glycine response, demonstrating that a protein binds this element, that binding is modulated by tetrahydrofolate, and that folate is required for the in vivo glycine response. In an heterologous CYC1 promoter the region needed for the glycine response of GCV2 (encoding the P-subunit of glycine decarboxylase) mediated repression that was relieved by glycine. It was also responsible for L-methionine control but not nitrogen repression. GCV1 and GCV3 have an homologous region in their promoters. The GCV1 region conferred a glycine response on an heterologous promoter acting as a repressor or activator depending on promoter context. A protein was identified that bound to the glycine regulatory regions of GCV1 and GCV2 only if the CTTCTT motif was intact. This protein protected a 17-base pair CATCN7CTTCTT region of GCV2 that is conserved between GCV1 and GCV2. Protein binding was increased by tetrahydrofolate, and use of a fol1 deletion mutant indicated the involvement of a folate in the in vivo glycine response. Tetrahydrofolate or a derivative may act as a ligand for the transcription factor controlling expression of one-carbon metabolism genes.

Topics: Amino Acid Oxidoreductases; Aminomethyltransferase; Base Sequence; Carrier Proteins; DNA Footprinting; DNA, Fungal; Gene Expression Regulation, Fungal; Glycine; Glycine Dehydrogenase (Decarboxylating); Hydroxymethyl and Formyl Transferases; Molecular Sequence Data; Multienzyme Complexes; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tetrahydrofolates

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

Topics: Amino Acid Oxidoreductases; Aminomethyltransferase; Carrier Proteins; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glycine; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Glycine Hydroxymethyltransferase; Hydroxymethyl and Formyl Transferases; Mitochondria; Multienzyme Complexes; NAD; Oxidation-Reduction; Plant Proteins; Plants; Pyruvate Dehydrogenase Complex; Tetrahydrofolates; Thioctic Acid; Transferases

T-protein, one of the components of the glycine cleavage system, catalyzes the synthesis of the H-protein-bound intermediate from methylenetetrahydrofolate, ammonia, and H-protein having a reduced lipoyl prosthetic group (Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J. Biol. Chem. 257, 135-139). Spectroscopic studies indicated that the utilization of methylenetetrahydrofolate occurred only in the presence of the three substrates, indicating the formation of a quaternary complex. The amount of methylenetetrahydrofolate consumed was equal to that of methylene carbon attached to H-protein. Steady-state kinetic studies show that the reaction proceeds through an Ordered Ter Bi mechanism. Reduced H-protein is the first substrate that binds T-protein followed by methylenetetrahydrofolate and ammonia. The order of release of products is tetrahydrofolate and the H-protein-bound intermediate. Km values for H-protein, methylenetetrahydrofolate, and ammonia are 0.55 microM, 0.32 mM, and 22 mM, respectively.

Topics: Amino Acid Oxidoreductases; Aminomethyltransferase; Ammonia; Ammonium Chloride; Carrier Proteins; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Hydroxymethyl and Formyl Transferases; Kinetics; Protein Binding; Tetrahydrofolates; Transferases

Topics: 5,10-Methylenetetrahydrofolate Reductase (FADH2); Acetaldehyde; Alcohol Oxidoreductases; Alcoholism; Aminomethyltransferase; Animals; Cattle; Chickens; Glycine Hydroxymethyltransferase; Hydroxymethyl and Formyl Transferases; In Vitro Techniques; Liver; Methylenetetrahydrofolate Dehydrogenase (NADP); Substrate Specificity; Tetrahydrofolates; Thymidylate Synthase; Transferases