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Functional characterization of GcpE, an essential enzyme of the non‐mevalonate pathway of isoprenoid biosynthesis
Author(s) -
Kollas Ann-Kristin,
Duin Evert C,
Eberl Matthias,
Altincicek Boran,
Hintz Martin,
Reichenberg Armin,
Henschker Dajana,
Henne Anke,
Steinbrecher Irina,
Ostrovsky Dmitry N,
Hedderich Reiner,
Beck Ewald,
Jomaa Hassan,
Wiesner Jochen
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03725-0
Subject(s) - biosynthesis , thermus thermophilus , mevalonate pathway , biochemistry , enzyme , terpenoid , escherichia coli , enzyme kinetics , biology , plastid , stereochemistry , chemistry , active site , gene , chloroplast
The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2‐ C ‐methyl‐ D ‐erythritol‐4‐phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid‐like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen‐free conditions. The protein was enzymatically active in converting 2‐ C ‐methyl‐ D ‐erythritol‐2,4‐cyclodiphosphate (MEcPP) into ( E )‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 μmol min −1 mg −1 at pH 7.5 and 55°C. The k cat value was 0.4 s −1 and the K m value for HMBPP 0.42 mM.