Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis

• Published in: FEBS letters 2002-12, Vol.532 (3), p.432-436
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate; 2-C-Methyl-D-erythritol-2,4-cyclodiphosphate; 2-C-Methyl-D-erythritol-4-phosphate pathway; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Chromatography, High Pressure Liquid; DMAPP, dimethylallyl diphosphate; Dose-Response Relationship, Drug; DOXP, 1-deoxy-D-xylulose-5-phosphate; Electrophoresis, Polyacrylamide Gel; Enzymes; Escherichia coli - metabolism; GcpE; HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate; IPP, isopentenyl diphosphate; Isoprenoid biosynthesis; Kinetics; Light; Lymphocyte Activation; LytB; MEcPP, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; Models, Chemical; Molecular Sequence Data; Organophosphates - pharmacology; Oxygen - metabolism; PBMC, peripheral blood mononuclear cells; Plasmids - metabolism; Plastids - metabolism; Polyisoprenyl Phosphates - biosynthesis; Pyrimidines - pharmacology; Recombinant Proteins - metabolism; Spectrometry, Mass, Electrospray Ionization; T-Lymphocytes - metabolism; Thermus thermophilus - metabolism; Ultraviolet Rays
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(02)03725-0

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 kcat value was 0.4 s−1 and the Km value for HMBPP 0.42 mM.