Macrolide antibiotic biosynthesis: isolation and properties of two forms of 6-deoxyerythronolide B hydroxylase from Saccharopolyspora erythraea (Streptomyces erythreus).
A cytochrome P-450 monooxygenase that catalyzes the hydroxylation of 6-deoxyerythronolide B, an intermediate of erythromycin A biosynthesis in Saccharopolyspora erythraea (formerly Streptomyces erythreus), was resolved into two forms, P-450I and P-450II, by hydroxylapatite chromatography. These two proteins were purified to homogeneity from the CA 340 strain and found to have a P-450 content of 17.5 and 15.2 nmol/mg of protein, respectively. Either enzyme catalyzed the NADPH-dependent hydroxylation of 6-deoxyerythronolide B and (9R)- or (9S)-9-deoxo-9-hydroxy-6-deoxyerythronolide B in vitro when reconstituted with other electron-transport components from S. erythraea. Both of them had a Mr of 44,220 +/- 1350, a pI of 4.6, similar amino acid compositions, and an identical N-terminal sequence for the first five amino acids. They also showed identical antigenicity and cross-reactivity against polyvalent and specific antibodies and contained cytochrome P-450 in the low spin state with absorption maxima at 416, 532, and 565 nm. Their distinguishing characteristics were different activities toward the (9S)-9-deoxo-9-hydroxy-6-deoxyerythronolide B substrate and slightly different absorbance maxima in their dithionite-reduced CO-complexed spectra.
D000596:Amino Acids / Q000032:analysis; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D004917:Erythromycin / Q000031:analogs & derivatives / Q000096:biosynthesis; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D000939:Epitopes; D007527:Isoenzymes; C034277:6-deoxyerythronolide B; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea