title: The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae.
abstract: The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 A) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 A)or narbomycin (resolution 1.7 A). A central feature of the enzyme-substrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3' dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.
authors.affiliation: Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA. davidhs@umich.edu
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
authors.forename: Liudmila V
authors.initials: LV
authors.lastname: Yermalitskaya
authors.forename: Youngchang
authors.initials: Y
authors.lastname: Kim
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Smith
authors.forename: Michael R
authors.initials: MR
authors.lastname: Waterman
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Podust
mesh_terms: D000606:Amino Sugars / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D000606:Amino Sugars; D001426:Bacterial Proteins; D008024:Ligands; D018942:Macrolides; C110125:desosamine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C442772:PikC cytochrome P450 hydroxylase