Analysis of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase

Notwithstanding the "one-module-one-elongation-cycle" 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. TOC/Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/426894v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@150bdbforg.highwire.dtl.DTLVardef@1d958b6org.highwire.dtl.DTLVardef@4f19c7org.highwire.dtl.DTLVardef@1256656_HPS_FORMAT_FIGEXP M_FIG C_FIG