title: Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway.
abstract: Cytochrome P450 monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing 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. In the present 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 experiments, steady-state kinetics studies, and X-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to 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.
authors.affiliation: Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: DeMars
authors.affiliation: Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California , San Diego, California 92093, United States.
authors.forename: Fang
authors.initials: F
authors.lastname: Sheng
authors.affiliation: Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Sung Ryeol
authors.initials: SR
authors.lastname: Park
authors.affiliation: Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Lowell
authors.affiliation: Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California , San Diego, California 92093, United States.
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Podust
authors.affiliation: Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.|Department of Medicinal Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.|Department of Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.|Department of Microbiology & Immunology, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006900:Hydroxylation; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: D018942:Macrolides; C026686:mycinamicins; D003577:Cytochrome P-450 Enzyme System