Cytochrome P450s are one of the most widely distributed classes of enzymes in nature, catalyzing the oxidation of a broad range of natural product and xenobiotic small molecules. Although hundreds of P450 hydroxylases have been examined in the oxidative metabolism of xenobiotics and steroids, only a small number have been studied in bacterial secondary metabolism, especially in macrolide antibiotic biosynthetic pathways. In most of these pathways, hydroxylation(s) occurs in the late stages of biosynthesis after formation of the macrolide by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of anti-infective activity. Thus, the creation of novel macrolide analogs through combinatorial biosynthesis and chemoenzymatic synthesis warrants a concomitant effort towards the development of macrolide monooxygenases with broad substrate specificity. The aim of the proposed work is to develop a thorough understanding of the substrate flexibility and functionality of the cytochrome P450-PikC macrolide monooxygenase. Our recent structure determination of the enzyme has provided fascinating new insights into its catalytic mechanism and ability to generate several products by hydroxylation of the 12-membered ring macrolide YC-17 and the 14-membered ring macrolide narbomycin. This information will direct protein engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze hydroxylation or epoxidation reactions. Moreover, we plan to investigate the unprecedented desosamine sugar-mediated anchoring of macrolides within the PikC binding domain to develop engineered monooxygenases with versatile substrate selectivity. Specific Aim 1. Determination of the PikC structure and the mechanism(s) of hydroxylation of narbomycin and YC-17. Specific Aim 2. Explore the role of macrolide sugar-mediated anchoring on substrate binding and catalytic activity of PikC. Specific Aim 3. Engineering of novel macrolide hydroxylases.