The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae

J Biol Chem. 2006 Sep 8;281(36):26289-97. doi: 10.1074/jbc.M605478200. Epub 2006 Jul 6.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Sugars / chemistry
  • Amino Sugars / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Crystallography, X-Ray
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Ligands
  • Macrolides / chemistry
  • Macrolides / metabolism
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary*
  • Streptomyces / enzymology*


  • Amino Sugars
  • Bacterial Proteins
  • Ligands
  • Macrolides
  • desosamine
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • PikC cytochrome P450 hydroxylase

Associated data

  • PDB/2BVJ
  • PDB/2C6H
  • PDB/2C7X