Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase–dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance

Manickam Yogavel,Joanne E Nettleship,Akansha Sharma,Karl Harlos,Abhishek Jamwal,Rini Chaturvedi,Manmohan Sharma,Vitul Jain,Jyoti Chhibber-Goel,Amit Sharma

doi:10.1074/jbc.ra118.004558

Abstract

The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.

Highlights

The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway
The overall fold of the PvHPPK–DHPS domains is similar to their known homologs
The PvHPPK–DHPS was crystallized in the presence of 6-hydroxymethylpterin-diphosphate (PtPP), pterin, the ATP analog AMPCPP, and pABA (Fig. 2A)

Summary

To whom correspondence should be addressed

DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites. Malaria remains a constant public health threat because of the emergence of drug-resistant strains across endemic regions [2]. Tetrahydrofolate is an essential cofactor that is vital for metabolic reactions involving one-carbon transfer [4]. Most notably, it is required for the synthesis of nucleic acid precursors like purines and thymidine and for methionine, glycine, and pantothenate [5]. The malaria parasite genomes encode fused 6-hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains (Fig. 1A) that perform sequential reactions wherein HPPK catalyzes transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (DHP) resulting in 6-hydroxymethyl-7,8-dihydropterinpyrophosphate (DHPPP) [8, 9]. DHPS acts as a crucial convergence point in the folate pathway and catalyzes the condensation of 4-aminobenzoic acid (pABA) and DHPPP to form the intermediate 7,8-dihydropteroate (Fig. 1B) [10]. 7,8-Dihydropteroate is fed into the folate synthesis path-

The abbreviations used are

Results

Discussion

Experimental procedures