Structure and Biochemistry of Cytochromes P450 Involved in the Biosynthesis of Macrolide Antibiotics

Selective functionalization of chemically inert carbon-hydrogen (C–H) bonds embodies one of the grand challenges of organic chemistry and provides a key focus of research in the field. C–H functionalization can provide a valuable means to improve the efficiency of complex molecule synthesis, but significant challenges remain with respect to achieving high site selectivity in the presence of multiple unique C–H bonds in a given target. As a complement to small-molecule transition metal-based catalysts, enzymes have received increasing attention in recent years as potential biocatalysts for carrying out selective C–H bond oxidation reactions. Members of the cytochrome P450 superfamily of monooxygenases (P450s) are some of nature’s most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize C–H bonds has led to their growing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. The work presented herein details studies that were carried out to biochemically and structurally characterize diverse bacterial P450s involved in the biosynthesis of 16-membered ring macrolide antibiotics with significant potential for development into robust biocatalysts for the late-stage functionalization of complex molecules. In an initial study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding and kinetics experiments along with X-ray crystallographic analysis of MycCI bound to its native substrate. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared with that of MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity. Next, we performed a more in-depth analysis of TylHI in an attempt to understand the molecular basis for its substrate specificity. Turnover and equilibrium binding experiments with substrate analogs revealed that this enzyme exhibits a strict preference for 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. These results were partially explained through analysis of the X-ray crystal structure of TylHI in complex with its native substrate together with biochemical characterization of several site-directed mutants. Comparative analysis of the MycCI/TylHI homolog ChmHI from the chalcomycin biosynthetic pathway provided a basis for constructing MycCI/TylHI chimeras in order to gain further insight into the features dictating the differences in the reactivity profiles of these two related P450s. These experiments unveiled the central role of the BC loop region in influencing the binding properties of 16-membered ring substrates to MycCI and TylHI. Finally, comparative analysis of several different P450s from the mycinamicin (MycCI), tylosin (TylI), and juvenimicin (JuvC and JuvD) biosynthetic pathways revealed unique substrate preferences and catalytic outcomes that facilitated their subsequent employment as biocatalysts in the chemoenzymatic synthesis of tylactone-based macrolide antibiotics.