Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum

Abstract

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.

Figure 1

The DHPS reaction and isotopically labeled pABA substrates used in the BIE analysis. (a) DHPS catalyzes the formation of DHP via condensation of DHPP with pABA. (b) BIE measurements were performed using [3,5-³H]pABA and [7-¹⁴C]pABA. DHPP, 6-hydroxymethyl-7,8-dihydropterin pyrophosphate; DHPS, dihydropteroate synthase; pABA, para-aminobenzoic acid; PP_i, pyrophosphate; DHP, dihydropteroate.

Figure 2

Calculated effects of changes in dielectric environment and geometric distortion on BIEs for [3,5-³H]pABA. (a) Equilibrium BIEs were calculated for pABA using water (ε = 78.4), methanol (ε = 32.6), acetone (ε = 20.5), dichloroethane (ε = 10.1), chlorobenzene (ε = 5.7), or carbon tetrachloride (ε = 2.2) as an implicit solvent model (PCM) in the bound state. The red line illustrates the experimental BIE value measured for SaDHPS. (b) Calculated effects of out-of-plane distortions of the C3–H3 and C5–H5 bonds. Restraints were imposed on the bound state by fixing the H3–C3–C4–C5 and H5–C5–C4–C3 dihedrals at increasing degrees from the equilibrium angle. The red line illustrates the experimental BIE value measured for SaDHPS. (c) Calculated effects of C3–H3 and C5–H5 bond length compression. The red line illustrates the experimental BIE value measured for PfDHPS.

Figure 3

Comparison of primary structures for the PfDHPS domain and SaDHPS. Sequences of the full length PfHPPK–DHPS and SaDHPS proteins were compared, and the resulting alignment was truncated to show only the DHPS domains. Strictly conserved residues are shown in blue. Red circles identify residues predicted to interact with substrates by homology to other DHPS variants. Nonhomologous, plasmodium-specific inserts are shown in green. The insert deleted from PfHPPK–DHPS in this study extends between residues 628 and 668. The sequence alignment was performed using COBALT.