Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thaliana

Abstract

The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate at a rate of 5.6 μmol·min -1·mg-1 (kcat 4.4 s-1). The Michaelis constants for 1-deoxy-d-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 μm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 μmol· min-1·mg-1. © 2006 The Authors.