Overproduction and characterization of the erythromycin C-12 hydroxylase, EryK.
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.
Lambalot
R H
RH
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Cane
D E
DE
Aparicio
J J
JJ
Katz
L
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004917:Erythromycin / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013053:Spectrophotometry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C078882:EryK protein, Saccharopolyspora erythraea