Properties of a “Split-and-Stuttering” Module of an Assembly Line Polyketide Synthase

Abstract

Notwithstanding the "one-module−one-elongationcycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all-trans-configured triene moiety in the polyketide product. Here, we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

Discovery and Biosynthesis of the Cytotoxic Polyene Terpenomycin in Human Pathogenic Nocardia

“…Unlike other known bafilomycin-like BGCs, the putative Ter PKS proteins include unusual features such as a "split" module (M9), iterative use of module 3, and skipping of module 2 (Figure 2). While there are several examples of cis-AT PKSs utilizing split and/or iterative modules, 41,42 including in Nocardia species, 43 the combination of all of these features in a single cis-AT PKS has not been previously reported.…”

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin

“… [32] To our knowledge there are only rare examples of iterative modules in trans ‐AT PKSs and to‐date these involve the assembly of conjugated alkene portions of the polyketide. [ 33 , 34 ]…”

“…The number of iterations performed may be guided by a combination of the elongation kinetics and noncovalent protein–protein interactions alongside a chain‐length gatekeeping role of the downstream KS. [33] Elegant experiments involving the expression of aurA , the gene encoding the iterating module of the aureothin PKS from Streptomyces thioluteus , in a heterologous host led to the observation of additional iterations (up to four) in the absence of gatekeeper control (the downstream KS in AurB). [36] For MmpF and MacpA, the MmpB_KS functions as a strict gatekeeper, recognising and transferring only the 7HH‐MacpA intermediate.…”

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin

“…The action of MmpF essentially resembles bacterial Type II fatty acid biosynthesis where a full reductive cycle of β‐keto processing occurs before the fatty acid is returned to a KS for the next elongation step (Figure 6A). The number of iterations performed may be guided by a combination of the elongation kinetics and noncovalent protein–protein interactions alongside a chain‐length gatekeeping role of the downstream KS [33] . Elegant experiments involving the expression of aurA , the gene encoding the iterating module of the aureothin PKS from Streptomyces thioluteus , in a heterologous host led to the observation of additional iterations (up to four) in the absence of gatekeeper control (the downstream KS in AurB) [36] .…”

“…[32] To our knowledge there are only rare examples of iterative modules in trans-AT PKSs and to-date these involve the assembly of conjugated alkene portions of the polyketide. [33,34] MmpF, derived from a degraded cis-AT KS-AT didomain represents a new example of a fully reducing stuttering module that has been acquired by a trans-AT system (Figure 6A). Uniquely this is not embedded in a larger multi-functional module but as a standalone unit that has certain interesting implications.…”