The druggable antimalarial target PfDXR : overproduction strategies and kinetic characterization
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Date
Authors
Goble, Jessica L.
Johnson, Hailey
De Ridder, Jaco
Stephens, Linda L.
Louw, Abraham Izak
Blatch, Gregory L.
Boshoff, Aileen
Journal Title
Journal ISSN
Volume Title
Publisher
Bentham Science Publisher
Abstract
Plasmodium falciparum 1–deoxy–D–xylulose–5–phosphate reductoisomerase (PfDXR) is a key enzyme in the
synthesis of isoprenoids in the malaria parasite, using a pathway that is absent in the human host. This enzyme is receiving
attention as it has been validated as a promising drug target. However, an impediment to the characterisation of this enzyme
has been the inability to obtain sufficient quantities of the enzyme in a soluble and functional form. The expression
of PfDXR from the codon harmonised coding region, under conditions of strongly controlled transcription and induction,
resulted in a yield of 2 – 4 mg/L of enzyme, which is 8 to 10–fold higher than previously reported yields. The kinetic parameters
Km, Vmax and kcat were determined for PfDXR using an NADPH–dependent assay. Residues K295 and K297,
unique to species of Plasmodium and located in the catalytic hatch region; and residues V114 and N115, essential for
NADPH binding, were mutated to resemble those found in E. coli DXR. Interestingly, these mutations decreased the substrate
affinity of PfDXR to values resembling that of E. coli DXR. PfDXR-K295N, K297S and PfDXR-V114A, N115G
demonstrated a decreased ability to turnover substrate by 4–fold and 2-fold respectively in comparison to PfDXR. This
study indicates a difference in the role of the catalytic hatch in capturing substrate by species of Plasmodium. The results
of this study could contribute to the development of inhibitors of PfDXR.
Description
Keywords
Plasmodium falciparum, DXR, Anti–malarial, Heterologous expression, Molecular chaperones
Sustainable Development Goals
Citation
Goble, JL, Johnson, H, De Ridder, J, Stephens, LL, Louw, A, Blatch, GL & Boshoff, A 2013, 'The druggable antimalarial target PfDXR : overproduction strategies and kinetic characterization', Protein and Peptide Letters, vol. 20, no. 2, pp.115-124.