Abstract
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.
Highlights
Serine hydroxymethyltransferase (SHMT or GlyA; EC 2.1.2.1) is a ubiquitous pyridoxal 5’phosphate (PLP)-dependent enzyme
As the translated polypeptide shows (e.g.) 53% sequence identity (72% sequence similarity) with E. coli SHMT (EcSHMT) and 68% sequence identity (80% sequence similarity) with the Campylobacter jejuni enzyme, open reading frame (ORF) HP0183 was predicted to code for a serine hydroxymethyltransferase
As there is no in silico evidence for the presence of a glycine cleavage system in H. pylori, we tested directly, whether the polypeptide encoded by HP0183 can functionally complement growth defects of an E. coli strain impaired in serine hydroxymethyltransferase activity
Summary
Serine hydroxymethyltransferase (SHMT or GlyA; EC 2.1.2.1) is a ubiquitous pyridoxal 5’phosphate (PLP)-dependent enzyme. In Eukarya and most bacterial species, SHMT is part of the thymidylate/folate cycle, together with canonical thymidylate synthase ThyA (EC 2.1.1.45) and dihydrofolate reductase DHFR (EC 1.5.1.3) [14]. The functional association of SHMT, ThyA and DHFR was thought to be a universally conserved evolutionary feature (Fig 1A). This view changed with the discovery of a novel family of flavin-dependent thymidylate synthases, ThyX, essential for de novo dTMP synthesis (EC 2.1.1.148) [15] (Fig 1B). On the basis of mathematical modeling of the bacterial folate metabolism, we have proposed that a very low dihydrofolate reductase activity, provided by moonlighting enzymes, is sufficient to rescue thymidylate synthesis in the presence of ThyX.
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