The uridylyltransferase GlnD and tRNA modification GTPase MnmE allosterically control Escherichia coli folylpoly-γ-glutamate synthase FolC

Abstract

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

One‐carbon metabolism, folate, zinc and translation

“…FolC is further regulated by the glutamate/glutamine‐sensing uridylyltransferase GlnD and UDP‐glucose dehydrogenase Ugd. FolC inhibits the GTPase activity of MnmE at low GTP concentrations, further tying up folic acid metabolism to tRNA modification (Rodionova et al , 2018).…”

“…However, while the modifications were likely important for proper integration of metabolism, they did not usually have a significant effect on the growth rate of E. coli strains in standard growth conditions. In contrast, when the host strain was deficient in the synthesis of polyamines, deletion of the mnmE or mnmG gene resulted in complete inhibition of growth unless the medium contained polyamines (Rodionova et al , 2018; Shalaeva et al , 2018; Gao et al , 2019; Keller et al , 2019).…”

A novel transcription factor PunR and Nac are involved in purine and purine nucleoside transporter punC regulation in E. coli

“…The PunR-producing strain was grown overnight, inoculated into a 50 ml culture, and induced with 0.6 mM IPTG after an OD 600 = 0.6 was reached. The cells were harvested after 4 hours and lysed as previously described (42). The puri ed protein and a 10 nM uorescently labelled DNA fragment (5'-AGG GGG-3') were incubated in the assay mixture.…”

UDP-glucose dehydrogenase Ugd inE. coliis activated by Gmd and RffD, is inhibited by CheY, and regulates swarming

“…Ugd, as well as the uridylyltransferase, GlnD, and the GTPase, MnmE, activate folylpoly-gamma-glutamate synthase, FolC in E. coli, by direct protein-protein interactions (6). Thus, Ugd appears to be part of a complex regulatory system, influencing and influenced by many types of physiological states and conditions.…”