E. coli MEP synthase

Steady-state kinetic analysis and substrate binding

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

Research output: Contribution to journalArticle

104 Citations (Scopus)

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
Externally publishedYes

Fingerprint

NADP
Escherichia coli
Phosphates
Kinetics
Substrates
Competitive Binding
Biosynthetic Pathways
Terpenes
Chromatography
2-C-methylerythritol 4-phosphate
1-deoxy-2-pentulose
deoxyxylulose phosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

E. coli MEP synthase : Steady-state kinetic analysis and substrate binding. / Koppisch, Andrew T; Fox, D. T.; Blagg, B. S J; Poulter, C. D.

In: Biochemistry, Vol. 41, No. 1, 08.01.2002, p. 236-243.

Research output: Contribution to journalArticle

Koppisch, Andrew T ; Fox, D. T. ; Blagg, B. S J ; Poulter, C. D. / E. coli MEP synthase : Steady-state kinetic analysis and substrate binding. In: Biochemistry. 2002 ; Vol. 41, No. 1. pp. 236-243.
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