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

J Biol Chem. 2018 Oct 5;293(40):15725-15732. doi: 10.1074/jbc.RA118.004425. Epub 2018 Aug 8.

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 Escherichia coli 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 in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the Khalf 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 in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

Keywords: FolC; G-protein MnmE; GTPase; GlnD; MnmE; Ugd; allosteric regulation; amino acid sensing; cytoplasmic GTP; enzyme kinetics; folate; folate metabolism; gram-negative bacteria; metabolism; tetrahydrofolate polyglutamylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Binding Sites
  • Enzyme Assays
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Folic Acid / biosynthesis
  • Folic Acid / chemistry
  • GTP Phosphohydrolases / chemistry*
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism
  • Gene Expression Regulation, Bacterial*
  • Glutamic Acid / chemistry
  • Glutamic Acid / metabolism
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism
  • Kinetics
  • Molecular Docking Simulation
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism
  • Peptide Synthases / chemistry*
  • Peptide Synthases / genetics
  • Peptide Synthases / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Pteroylpolyglutamic Acids / biosynthesis
  • Pteroylpolyglutamic Acids / chemistry
  • RNA, Transfer / chemistry
  • RNA, Transfer / metabolism
  • Substrate Specificity
  • Thermodynamics
  • Uridine Diphosphate Glucose Dehydrogenase / chemistry*
  • Uridine Diphosphate Glucose Dehydrogenase / genetics
  • Uridine Diphosphate Glucose Dehydrogenase / metabolism

Substances

  • Escherichia coli Proteins
  • Multienzyme Complexes
  • Pteroylpolyglutamic Acids
  • Glutamic Acid
  • Guanosine Triphosphate
  • RNA, Transfer
  • Folic Acid
  • Uridine Diphosphate Glucose Dehydrogenase
  • Nucleotidyltransferases
  • GTP Phosphohydrolases
  • MnmE protein, E coli
  • Peptide Synthases
  • folC protein, E coli