ABSTRACT
GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. The docking models, in conjunction with the results of the enzymatic assay with substrate analogues and site-directed mutagenesis suggested two distinct substrate binding modes for epoxidation and hydroxylation reactions, which explained how GfsF regulates the order of two oxidative reactions. These findings provide new insights into the reaction mechanism of multifunctional P450 monooxygenases.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Macrolides/metabolism , Cytochrome P-450 Enzyme System/analysis , Cytochrome P-450 Enzyme System/genetics , Hydrophobic and Hydrophilic Interactions , Macrolides/chemistry , Molecular Conformation , Molecular Docking Simulation , Mutagenesis, Site-Directed , Substrate SpecificityABSTRACT
Unified synthesis of FD-891 analogs and their structure-activity relationship are described. By using stereoselective allylation/crotylation and Evans aldol chemistry, six side-chain fragments having different length and terminus were synthesized. These fragments were coupled with a macrolactone fragment, improved synthesis of which was also developed here, to generate FD-891 and five truncated analogs. These synthetic compounds as well as three analogs obtained from fermentation of gene-disrupted Streptomyces graminofaciens mutants were tested for in vitro cytotoxic activity against HeLa cells. As a result, coexistence of the C8-C9 epoxide and side-chain terminus was found to be critical for the cytotoxic activity.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Macrolides/chemistry , Macrolides/pharmacology , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Epoxy Compounds/chemistry , HeLa Cells , Humans , Macrolides/chemical synthesis , Streptomyces/genetics , Streptomyces/metabolism , Structure-Activity RelationshipABSTRACT
A concise and unified strategy for the synthesis of C1-C18 macrolactone fragments of FD-891 and FD-892 as well as their analogues is reported. The strategy includes a stereoselective vinylogous Mukaiyama aldol reaction (VMAR) using chiral silyl ketene N,O-acetal to construct C6-C7 stereocenters, an E-selective ring closing metathesis to construct a C12-C13 olefin, and stereodivergent construction of a C8-C9 epoxide.
