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.