The 6-deoxyerythronolide B hydroxylase (EryF) is a soluble cytochrome P450 responsible for the stereospecific C-6 hydroxylation of the erythromycin precursor, 6-deoxyerythronolide B. Using the expression of the eryF gene in Escherichia coli [Andersen, J. F., & Hutchinson, C. R. (1992) J. Bacteriol. 174, 725-735] as the enzyme source, we examined the catalytic activity of the EryF protein toward several macrolide substrates related to 6-deoxyerythronolide B. The results of these studies were compared with measurements of the apparent dissociation constants for various substrates and with information from molecular modeling studies of the substrates and the enzyme-substrate complex. Only minor changes in the structure of 6-deoxyerythronolide B resulted in substrates with catalytic rates less than 1% of those seen with the natural substrate. Although the 9S epimer of 9-deoxo-9-hydroxy-6-deoxyerythronolide B was hydroxylated at a rate approximately equal to the natural substrate, the 9R epimer was hydroxylated at a 2-fold lower rate. Examination of molecular models revealed that the position of the 9-hydroxyl oxygen in the 9S epimer resembles that of the 9-oxo oxygen in the natural substrate more closely than in the 9R epimer. 8,8a-Deoxyoleandolide, which is identical to 6-deoxyerythronolide B except for the presence of a C-13 methyl group, and its (9S)-9-deoxo-9-hydroxy derivative were C-6 hydroxylated at a 4-fold lower rate than the natural substrate, and the 9-oxo form showed a substantially larger apparent dissociation constant.(ABSTRACT TRUNCATED AT 250 WORDS)