Mutagenesis of Folylpolyglutamate Synthetase Indicates That Dihydropteroate and Tetrahydrofolate Bind to the Same Site

Abstract

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.

Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery

“…The heterocycle of the pterin is secluded within a cavity and stacks between hydrophobic residues including Phe, Ala, Ile, and Leu. When the DHFS and FPGS activity is shared within a single protein, there is a separate inhibitory profile for each of the two enzymatic specificities [ 53 , 108 , 115 ]. The FPGS substrate contains additional functional groups on the pterin that require a larger binding pocket than what is found in DHFS enzymes.…”

Structures ofMycobacterium tuberculosisfolylpolyglutamate synthase complexed with ADP and AMPPCP

“…This in turn stabilizes the DHP loop through hydrophobic stacking interactions mediated by Tyr206 and facilitates stable binding of the DHP substrate. There is evidence from mutagenesis and binding studies that this site, formed by closure of the DHP loop, accommodates both DHP and monoglutamylated folate derivatives (Sheng et al, 2008) Surface representation of MtFPGS (a) and EcFPGS (b), showing the DHP-binding site, with DHPP from the EcFPGS structure modelled into the MtFPGS structure. The N-and C-terminal domains of MtFPGS are coloured teal and light teal, respectively, and those of EcFPGS green and light green, respectively.…”

Concentration-Dependent Processivity of Multiple Glutamate Ligations Catalyzed by Folylpoly-γ-glutamate Synthetase

“…Once the heterocycle is bound at the second site, the C-terminus of the DDAH 4 PteGlu 2 product would rearrange to position the γ-carboxyl of the terminal glutamate in close proximity to ATP for a second glutamate ligation reaction via the acyl phosphate intermediate (18). Subsequent multiple glutamate ligation reactions would occur with the heterocycle bound at the second site which is postulated to bind preferentially the polyglutamate derivatives (40, 43). A similar translocation of the electrophilic substrate, glutathione (GSH), is postulated in the reaction catalyzed by glutathionylspermidine synthetase (GspS, EC 6.3.1.8) based on crystallographic evidence; i.e., the position of GSH in the dead-end complex, GspS · GSH · ADP vs. the GSH portion of a phosphorylated bi-substrate phosphinic acid-containing pseudopeptide inhibitor (I) in the complex, GspS · I · ADP (44).…”