Abstract
Hydroxylation of C-12 is one of the final steps in the biosynthesis of erythromycin A (ErA). A point of uncertainty in the erythromycin pathway has been whether the C-12 hydroxylase operates on each of two possible substrates, erythromycin B (ErB) and erythromycin D (ErD). Stassi et al. have cloned the gene, designated eryK, which encodes the P-450 monooxygenase responsible for erythromycin C-12 hydroxylation in Saccharopolyspora erythraea [Stassi, D., Donadio, S., Staver, M. J., & Katz, L. (1993) J. Bacteriol. 175, 182-189]. We report the overproduction of EryK in Escherichia coli as insoluble inclusion bodies; the solubilization, refolding, and reconstitution of active holo-EryK; and kinetic confirmation of a 1200-1900-fold preference of the enzyme for ErD over the alternative C-12 hydroxylase substrate ErB. Our results indicate that ErB is a shunt metabolite in the erythromycin biosynthetic pathway.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Bacterial Proteins*
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Base Sequence
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Chromatography, High Pressure Liquid
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Cloning, Molecular
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Cytochrome P-450 Enzyme System / chemistry
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Cytochrome P-450 Enzyme System / genetics
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Cytochrome P-450 Enzyme System / metabolism*
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Erythromycin / biosynthesis
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Erythromycin / chemistry
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Gene Expression*
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Kinetics
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Mixed Function Oxygenases / chemistry
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Mixed Function Oxygenases / genetics
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Mixed Function Oxygenases / metabolism*
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Molecular Sequence Data
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Molecular Structure
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Polymerase Chain Reaction
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Protein Folding
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Solubility
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Spectrophotometry
Substances
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Bacterial Proteins
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Recombinant Proteins
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Erythromycin
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Cytochrome P-450 Enzyme System
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Mixed Function Oxygenases
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EryK protein, Saccharopolyspora erythraea