Abstract
Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal specific activity of 6.6+/-0.3 micromol x min(-1) x mg(-1) protein was determined at pH 7.5 and 60 degrees C. The k(cat) value of the LytB protein was 3.7+/-0.2 s(-1) and the K(m) value for HMBPP was 590+/-60 microM.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Bacterial Proteins / physiology*
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Catalysis
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Chromatography, High Pressure Liquid
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Cloning, Molecular
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Dose-Response Relationship, Drug
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Electrophoresis, Polyacrylamide Gel
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Erythritol / analogs & derivatives*
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Erythritol / metabolism*
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Escherichia coli / metabolism
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Escherichia coli Proteins*
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Eubacterium / genetics
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Eubacterium / metabolism
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Hydrogen-Ion Concentration
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Kinetics
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Models, Chemical
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Molecular Sequence Data
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Organophosphates / pharmacology
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Oxidoreductases / genetics
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Oxidoreductases / metabolism
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Oxidoreductases / physiology*
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Protein Binding
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Protein Structure, Tertiary
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Recombinant Proteins / metabolism
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Spectrometry, Mass, Electrospray Ionization
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Sugar Phosphates / metabolism*
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Temperature
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Time Factors
Substances
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2-C-methylerythritol 4-phosphate
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4-hydroxy-3-methyl-2-butenyl diphosphate
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Bacterial Proteins
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Escherichia coli Proteins
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Organophosphates
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Recombinant Proteins
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Sugar Phosphates
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Oxidoreductases
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ispH protein, E coli
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Erythritol