Comparative methods for recombinant production and characterization of 1-deoxy-D-xylulose-5-phosphate synthase from bacterial and apicomplexan sources.

Abstract

1-Deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the first step of the methylerythritol phosphate (MEP) pathway, which is required for isoprenoid biosynthesis in bacteria and apicomplexan parasites but is absent in humans. This pathway represents an important target for drug development, necessitating methods for recombinant production and biochemical characterization of DXPS from diverse organisms. This chapter describes a comparative framework for the cloning, expression, purification, and characterization of DXPS from the bacterium Deinococcus radiodurans and the apicomplexan Plasmodium vivax and Plasmodium falciparum. Strategies for construct design are presented, including systematic truncation of N-terminal targeting sequences required to obtain active enzymes. Expression optimization is detailed, along with chromatography workflows that preserve cofactor-dependent stability. Biochemical characterization was performed using a continuous spectrophotometric assay based on a coupled DXPS-DXR system. In addition, computational methodologies are presented, including molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) approaches for modeling thiamine diphosphate (ThDP)-dependent catalysis and active site electrostatics. Collectively, these methods provide a comprehensive and generalizable platform for the production, analysis, and mechanistic investigation of DXPS and related ThDP-dependent enzymes.