ABSTRACT
Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Micromonospora/enzymology , Polyketide Synthases/chemistry , Polyketide Synthases/metabolism , Acyl Carrier Protein/metabolism , Animals , Biosynthetic Pathways , Coenzyme A/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Iodine/metabolism , Ligands , Micromonospora/genetics , Micromonospora/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Protein Binding , Protein Structure, Tertiary , Resorcinols/metabolism , Streptomyces/geneticsABSTRACT
As the first in vitro characterization of a sugar N-oxidase, this study establishes CalE10 as the key oxidase involved in calicheamicin hydroxylamino glycoside formation. This study confirms that oxidation occurs at the sugar nucleotide stage prior to glycosyltransfer, and substrate specificity studies reveal CalE10-catalyzed oxidation to be regiospecific and to present trace amounts of the corresponding nitrosugar in vitro. This work also sets a precedent for the future study of other N-oxidases involved in hydroxylamino-, nitroso-, and/or nitrosugar formation.