Production of recombinant 1-deoxy-d-xylulose-5-phosphate synthase from Plasmodium vivax in Escherichia coli

Sumit Handa,Divya Ramamoorthy,Tyler J Spradling,Wayne C Guida,John H Adams,Kestutis G Bendinskas,David J Merkler

doi:10.1016/j.fob.2013.01.007

Sumit Handa, Divya Ramamoorthy + Show 5 more

Open Access

https://doi.org/10.1016/j.fob.2013.01.007

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Abstract

Humanity is burdened by malaria as millions are infected with this disease. Although advancements have been made in the treatment of malaria, optimism regarding our fight against malaria must be tempered against the problem of drug resistance in the Plasmodium parasites causing malaria. New targets are required to overcome the resistance problem. The enzymes of the mevalonate-independent pathway of isoprenoid biosynthesis are targets for the development of novel antimalarial drugs. One enzyme in this pathway, 1-deoxy-d-xylulose-5-phosphate synthase (DXS), catalyzes the conversion of 1-deoxy-d-xylulose-5-phosphate to isopentenylpyrophosphate and dimethylallyl phosphate. We demonstrate the use of a step deletion method to identify and eliminate the putative nuclear-encoded and transit peptides from full length DXS to yield a truncated, active, and soluble form of Plasmodium vivax DXS, the DXS catalytic core (DXScc).

Highlights

Malaria is a devastating health problem: approximately 50% of the world’s population is at risk of malaria, 216 million cases of malaria were reported in 2010, and over 650,000 people died of malaria in 2010 [1,2]
The enzymes of the mevalonateindependent pathway (MEP) are located in lumen of the apicoplast [29]; such enzymes from Plasmodium are difficult to express in E. coli
Only two MEP enzymes from Plasmodium have been expressed in E. coli: DXR [30] and 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase [31]

Summary

Introduction

Malaria is a devastating health problem: approximately 50% of the world’s population is at risk of malaria, 216 million cases of malaria were reported in 2010, and over 650,000 people died of malaria in 2010 [1,2]. Enzymes involved in type II fatty acid biosynthesis [12], isoprenoid biosynthesis [13], hemozoin biosynthesis [14], and iron sulfur cluster assembly [15] have been identified as potential targets for the development of novel antimalarial drugs. Of these pathways, the enzymes required for biosynthesis of the isoprenoid precursors, isopentenylpyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are attractive because the plasmodial route to IPP and DMAPP is completely orthogonal to the mammalian biosynthetic route. DXS catalyzes the condensation of pyruvate and d-glyceraldehyde-3phosphate to 1-deoxy-d-xylulose-5-phosphate (DXP) and CO2; this reaction being the first- and rate-determining step of MEP [22,23,24]. We find that the steady-state kinetic parameters and other biochemical features of P. vivax DXScc are consistent with data published for other DXS proteins [25,26]

Materials and methods

DXS and DXR purification by Ni-NTA affinity chromatography

Results