Dataset Information

Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria.

ABSTRACT: The aim of the present work was to further determine how the T-protein of the glycine-cleavage system and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the glycine-cleavage system was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.

SUBMITTER: Rebeille F

PROVIDER: S-EPMC1137213 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT.

Dataset Information

Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets