Abstract
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 micromol x min(-1) x mg(-1) at pH 7.5 and 55 degrees C. The kcat value was 0.4 s(-1) and the K(m) value for HMBPP 0.42 mM.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacterial Proteins / chemistry*
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Bacterial Proteins / metabolism*
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Chromatography, High Pressure Liquid
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Dose-Response Relationship, Drug
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Electrophoresis, Polyacrylamide Gel
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Enzymes*
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Escherichia coli / metabolism
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Kinetics
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Light
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Lymphocyte Activation
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Models, Chemical
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Molecular Sequence Data
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Organophosphates / pharmacology
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Oxygen / metabolism
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Plasmids / metabolism
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Plastids / metabolism
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Polyisoprenyl Phosphates / biosynthesis*
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Pyrimidines / pharmacology
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Recombinant Proteins / metabolism
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Spectrometry, Mass, Electrospray Ionization
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T-Lymphocytes / metabolism
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Thermus thermophilus / metabolism
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Ultraviolet Rays
Substances
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4-hydroxy-3-methyl-2-butenyl diphosphate
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Bacterial Proteins
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Enzymes
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Organophosphates
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Polyisoprenyl Phosphates
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Pyrimidines
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Recombinant Proteins
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hydroxymethylbutenyl 4-diphosphate synthase
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2-methyl-5,6-cyclopentapyrimidine
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Oxygen