Structural and Biochemical Investigation of Methylation and Elucidation of t-Butyl Formation in Polyketide Biosynthesis

Abstract

Polyketide natural products are a chemically diverse class of small molecules possessing a variety of therapeutic applications. Modular type I polyketide synthases (PKS) use a series of multienzyme modules in the assembly-line synthesis of polyketides from coenzyme A (CoA) building blocks. Biosynthetic intermediates are covalently linked to modules through an acyl carrier protein (ACP). The chemical diversity of polyketides is achieved through the variety of the catalytic domains within each module. In order to harness the biocatalytic power of PKS for the production of novel molecules, it is essential to understand the structural and mechanistic details of each biosynthetic tool. Through the use of x-ray crystallography and biochemical assays, this thesis investigates the role of methyltransferases (MTs), the least studied modification domain, in polyketide biosynthesis. Four distinct methyltransferases are found in PKS pathways: carbon (C-) and oxygen (O-) MTs occur in polyketide extension modules; two other MT types (MTL and MT2L) are exclusive to “loading” modules, which initiate PKS biosynthesis. Biochemical studies divulged the substrate for the C-MT, the first C-MT crystal structure revealed its common ancestry with the vestigial pseudo-MT of metazoan fatty acid synthase (mFAS). The PKS C-MTs and O-MTs were found to arise from different branches of the MT superfamily. Identification of essential catalytic residues for C-MTs and O-MTs provides insight into the methylation mechanism. A new biosynthetic route to a t-butyl group was a major discovery of this thesis. MTL and MT2L are associated with initiation modules that also contain GNAT-like acyltransferase/decarboxylase domains. Branched chain propionyl and isobutyryl starter units are generated by modules containing MTL, whereas MTL and MT2L together synthesize a t-butyl group. The AprA MTL from the apratoxin A biosynthetic pathway was discovered to be a rare iron-dependent MT, which converts malonyl-ACP to dimethylmalonyl-ACP through a methylmalonyl-ACP intermediate. In contrast, the AprA MT2L, a homolog of PKS C-MT domains, is a bifunctional enzyme that catalyzes coupled decarboxylation and methylation reactions to directly convert dimethylmalonyl-ACP to the t-butyl-containing pivaloyl-ACP. The AprA module was further visualized by negative-stain electron microscopy, revealing a dynamic module that may exist in different states for the MTL and MT2L catalytic steps. Analysis of MTL prompted further investigation of the GNAT-like domains in propionyl- and isobutyryl-ACP producing modules. Characterization of the GphF GNAT from the gephyronic acid biosynthetic pathway, which produces isobutyryl-ACP, demonstrated that the GNAT domain acts as a gatekeeper, selectively decarboxylating the MTL methylation product (dimethylmalonyl-ACP) for further processing by the enzymatic assembly line. Surprisingly, the expected acyl transfer activity was not detected, prompting the reclassification of PKS GNAT-like domains as acyl-ACP decarboxylases. The bacterial FAS malonyl-acyltransferase was investigated as a candidate for the acyltransfer reaction. The FAS malonyl-acyltransferase supports the initial acyl transfer step to prime the loading module ACP, potentially linking primary and secondary metabolism in the producing organism. Characterization of PKS MTs and the acyl-ACP decarboxylases advances our understanding of the biosynthesis of many valuable natural products and provides initial tools for the development of biocatalysts capable of synthetically challenging stereo- and regiospecific methylation reactions.