Purification and reconstitution of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis.
The hydroxylation of 6-deoxyerythronolide B (6D) to erythronolide B, a step in the biosynthesis of the 14-membered macrolide antibiotic erythromycin A by Saccharopolyspora erythraea, is catalyzed by a cytochrome P-450 monooxygenase that requires two electron transport proteins for the function of this terminal hydroxylase (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210, 1987). Two flavoproteins and an iron-sulfur protein (erythrodoxin) were purified from S. erythraea CA340 and shown to act with 6D hydroxylase to catalyze the hydroxylation of (9R)-[9-3H]9-deoxo-9-hydroxy-6D in vitro in a suitably reconstituted system. The flavoproteins contained flavin adenine dinucleotide and exhibited characteristic absorption maxima at 356 and 456 nm. The one with an Mr of 47,000 showed NADPH-dependent diaphorase and cytochrome c reductase activity, and the other, with an Mr of 53,000 showed NADH-dependent activities of the same two types. Erythrodoxin contained acid-labile sulfur and iron, had an Mr of 27,500, and showed a broad absorption maximum between 394 and 404 nm. The sequence of its first 15 amino acids, except for position 12, was the same as that of the ferredoxin from Mycobacterium smegmatis.
D001426:Bacterial Proteins; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004917:Erythromycin / Q000096:biosynthesis*; D005288:Ferredoxins / Q000302:isolation & purification; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009245:NADH Dehydrogenase / Q000302:isolation & purification; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010105:Oxygenases / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism
D001426:Bacterial Proteins; D005288:Ferredoxins; D005420:Flavoproteins; C055445:erythrodoxin; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D009251:NADPH-Ferrihemoprotein Reductase; D009245:NADH Dehydrogenase