Identifying Structural Determinants of Product Specificity in Leishmania major Farnesyl Diphosphate Synthase

Abstract

Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C₅) with isopentenyl diphosphate (IPP; C₅) and the resulting geranyl diphosphate (GPP; C₁₀) with another molecule of IPP, eventually producing farnesyl diphosphate (FPP; C₁₅), which is a precursor for the biosynthesis of a vast majority of isoprenoids. Previous studies of FPPS have highlighted the importance of the structure around the hydrophobic chain elongation path in determining product specificity. To investigate what structural features define the final chain length of the product in FPPS from Leishmania major, we designed and expressed six mutants of LmFPPS by replacing small amino acids around the binding pocket with bulky residues. Using enzymatic assays, binding kinetics, and crystallographic studies, we analyzed the effects of these mutations on the activity and product specificity of FPPS. Our results revealed that replacement of Thr-164 with tryptophan and phenylalanine completely abolished the activity of FPPS. Intriguingly, the T164Y substitution displayed dual product specificity and produced a mixture GPP and FPP as final products, with an activity for FPP synthesis that was lower than that of the wild-type enzyme. These data indicate that Thr-164 is a potential regulator of product specificity.

Figure 1.. Structural basis of site-directed mutations in LmFPPS.

(A) Active site of WT-LmFPPS (PDB ID: 4JZX)in complex with bisphosphonate inhibitor 476A (grey chain; colored by atoms) and substrate IPP(colored by atoms). The amino acid residues mutated in this study, E97 and T164 are illustrated as sticks. (B) Surface representation of the binding pocket of LmFPPS showing 476A and IPP buried in the allylic and homoallylic sites respectively.

Figure 2.. Purified recombinant WT-LmFPPS and mutants.

SDS-PAGE analysis of purified LmFPPS proteins. WT-LmFPPS and six mutants are shown(~40 kDa).

Figure 3.. DSF analyses of WT-LmFPPS and the mutants.

(A) Melt curves for the WT enzyme (shown in blue) and mutants, T164F-LmFPPS, T164Y-LmFPPS and T164W-LmFPPS (shown in orange, grey and yellow respectively) in Tris buffer, pH 8. Data are represented in relative fluorescence units (RFU) versus temperature. (B) The negative derivative of RFU versus temperature. The melting temperature ‘Tm’ corresponds to the peak or maximum of the first negative derivative of the curve.

Figure 4.. T164Y-LmFPPS produces both GPP and FPP as revealed by radioactive assay.

TLC autoradiochromatogram showing the activity of WT and mutant LmFPPSs. The spots for standard alcohols, geraniol and farnesol are shown in the last two lanes.

Figure 5.. Binding of FPP and GPP to WT and mutant LmFPPSs determined by SPR.

Steady-state dose response curves for binding of FPP to (A) WT-LmFPPS and (B) E97Y-LmFPPS. FPP was loaded in increasing concentrations at 1.5, 6.2, 25 and 100 μM. Dotted vertical grey lines indicate the values of steady-state affinity ‘K_D’. Sensorgrams for multi-cycle kinetics are depicted as inserts. Binding of GPP to (C) WT-LmFPPS and (D) E97Y-LmFPPS. GPP was loaded in increasing concentrations at 0.1, 0.39 and 1.5 μM (C) and at 0.1, 0.39, 1.5, 6.2 and 25 μM (D).All SPR experiments were performed two times.

Figure 6.. Structural insights into LmFPPS mutants.

Active sites of (A) WT-LmFPPS (PDB ID: 4JZX) (shown in green). (B) E97Y-LmFPPS (shown in blue), (C) T164W-LmFPPS (shown in magenta) and (D) T164Y-LmFPPS (shown in cyan) structures. The mutant structures are aligned with the WT-LmFPPS structure. Bound ligands, inhibitor 476A (grey chain; colored by atoms) and substrate IPP (colored by atoms) are shown in all the structures. Product FPP (pink chain; colored by atoms) was modeled from avian FPPS structure (PDB ID: 1UBX). The WT residues (E97 and T164) and the mutations (E97Y, T164W and T164Y) are illustrated as sticks.

Scheme 1.. Chain elongation reaction of FPPS.

FPPS catalyzes the consecutive condensation of dimethylallyl diphosphate (DMAPP) with isopentenyl diphosphate (IPP) and the resulting geranyl diphosphate (GPP) with another molecule of IPP to form farnesyl diphosphate (FPP).