E. coli MEP synthase: Steady-state kinetic analysis and substrate binding

A. T. Koppisch, D. T. Fox, B. S.J. Blagg, C. D. Poulter

Research output: Contribution to journalArticle

110 Scopus citations

Abstract

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: kcat = 116 ± 8 s-1, KMDXP = 115 ± 25 μM, and KMNADPH = 0.5 ± 0.2 μM. The rearrangement/reduction is reversible; Keq = 45 ± 6 for DXP and MEP at 150 μM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.

Original languageEnglish (US)
Pages (from-to)236-243
Number of pages8
JournalBiochemistry
Volume41
Issue number1
DOIs
StatePublished - Jan 8 2002

ASJC Scopus subject areas

  • Biochemistry

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