Abstract
In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.
Highlights
Folate molecules are used as cofactors in various metabolic pathways
Three-dimensional Structure of E. coli FolC—The 25% identity in sequence between E. coli FolC and L. casei folylpolyglutamate synthetase (FPGS) was found at the three-dimensional level
The proteins share the same overall structure. Both individual N-terminal and C-terminal domains of E. coli FolC superimposed with those of L. casei FPGS with a root mean square fit of 1.6 Å for the N-terminal domains and 1.9 Å for the C-terminal domains, their relative orientations are different in these two structures. (Fig. 2a)
Summary
The resulting plasmid, pVRC1432, was transferred into the E. coli BL21 (DE3) strain. This strain was grown at 37 °C in 1 liter of Luria-Bertani medium in presence of 50 mg/liter kanamycin. The supernatant (50 ml) was loaded on a Q-Sepharose Hi Load 16/10 column (Amersham Biosciences) and equilibrated with 50 mM Tris-HCl buffer, pH 7. The fraction containing the larger amount of the FolC protein was applied to a Superdex 75 16/60 column (Amersham Biosciences) and equilibrated with 50 mM Tris-HCl, 150 mM NaCl, pH 7. 2-ml fractions were collected at a flow rate of 1 ml/min. Using this protocol, 95 mg of pure FolC protein was obtained. Biochemical Data—The enzyme was incubated in 10 mM glycine-OH, 10 mM MgSO4, 50 mM KCl, pH 9.5, with DHP for measurement of DHFS
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