In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-β-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel “modularly iterative” mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.
Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of natural products. Mycobacterium tuberculosis (Mtb) exploits these polyketide biosynthetic enzymes to synthesize complex lipids, many of which are essential for its virulence. PKSs utilize two common mechanistic themes to produce these metabolites: (1) modular—in which each set of catalytic sites is used only once during the entire biosynthetic process and (2) iterative—in which the same set of active sites is used repeatedly. Our study with PKS12 protein from Mtb (the largest protein in this genome) reveals a third mechanism for polyketide biosynthesis. In this hybrid “modularly iterative” mechanism, PKS12 protein forms a supramolecular assembly to perform repetitive cycles of iterations. The protein assembly is formed by specific intermolecular interactions between N- and C-terminus linkers, analogous to modular PKSs. Our study adds a new dimension to the existing catalytic and mechanistic versatility of PKSs, providing a new perspective on how metabolic diversity could be generated by different combinations of existing functional scaffolds.
A novel iterative biosynthetic mechanism for multifunctional polyketide synthases reveals how the metabolic diversity of this enzyme family can arise by using existing scaffolds in novel combinations.