tailieunhanh - Báo cáo khoa học: Isoprenoid biosynthesis in plants – 2C-methyl-D-erythritol4-phosphate synthase (IspC protein) of Arabidopsis thaliana

TheispC gene of Arabidopsis thalianawas expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia colistrain. 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 )1 Æmg )1 (kcat s )1 ). The Michaelis constants for 1-deoxy-d-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30lm, respectively. . | ễFEBS Journal Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase IspC protein of Arabidopsis thaliana Felix Rohdich1 Susan Lauw1 Johannes Kaiser1 Richard Feicht1 Peter Kohler2 Adelbert Bacher1 and Wolfgang Eisenreich1 1 Lehrstuhl fur Organische Chemie und Biochemie Technische Universitat Munchen Garching Germany 2 Deutsche Forschungsanstalt fur Lebensmittelchemie Garching Germany Keywords deoxyxylulose dimethylallyl diphosphate herbicide isopentenyl diphosphate terpene Correspondence F. Rohdich and W. Eisenreich Lehrstuhl fur Organische Chemie und Biochemie Technische Universitat Munchen Lichtenbergstr. 4 D-85747 Garching Germany Fax 49 89 289 13363 Tel 49 89 289 13336 E-mail 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 pmol-min-1-mg-1 kcat s-1 . The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 pM 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 . 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 pmol-min 1-mg 1. Received 7 April2006 revised 7 July 2006 accepted 2 August 2006 doi The recently discovered nonmevalonate pathway of isoprenoid biosynthesis has been shown to afford the building blocks isopentenyl diphosphate and dimethylallyl diphosphate for a large number of plant terpenes that are biosynthesized in the plastid compartment. On the other hand small amounts of terpenes .