Assembly line polyketide synthases (PKSs) are fascinating biological machines that catalyze vectorial polyketide biosynthesis, namely the ability to channel a growing polyketide chain through a uniquely defined sequence of acyl carrier protein (ACP) and ketosynthase (KS) domains via alternating chain translocation and elongation reactions. The 6-deoxyerythronolide B synthase is a prototypical example, with 22 distinct enzymes distributed over three large homodimeric proteins. We seek to understand the mechanisms enabling vectorial biosynthesis along with those that diverge rapidly to spawn new biosynthetic pathways. We also seek to exploit this knowledge for the design of chimeric PKSs with catalytic activity that compares well to their naturally occurring counterparts. To these ends, the following Specific Aims are proposed: 1) Structural studies: We seek to solve the structures of a PKS module (or its catalytic core, comprised of its KS and acyltransferase (AT) domains along with flanking linkers) in states that can unequivocally be associated with either chain translocation or chain elongation, and to visualize how the KS-AT fragment interacts with its ACP partner in each of these states. Key tools already established for this purpose are: (i) Methods to crystallize the catalytic cores of two PKS modules; (ii) A method to crosslink the catalytic core of a module to either ACP partner; and (iii) High-affinity Fab antibodies that bind distinct domains of intact modules and stabilize their presumably dynamic conformations without inhibiting catalysis. 2) Engineering chimeric PKSs: Turnover of chimeric PKSs derived by linking intact modules from heterologous sources is invariably poor, principally due to suboptimal ACP-KS recognition at the chimeric junction. To solve this problem, we will develop streamlined methods to (i) identify heterologous module pairs that interface well with each other; and (ii) improve turnover of a given chimera by tuning ACP-KS interactions. Key tools already established for this purpose are (i) a panel of chimeric PKSs showing weak but measurable turnover; (ii) phage-display for selected ACPs; and (ii) identification of a specific helix in donor ACPs that promotes ACP ? KS chain translocation. 3) Dissecting the turnstile mechanism: We have observed that vectorial polyketide biosynthesis is synchronized by a ?turnstile? mechanism that energetically couples elongation of the growing polyketide chain to its intermodular translocation. Our working hypothesis is that the turnstile serves two important roles: (i) It prevents ?stuttering? (back-transfer) of a newly elongated polyketide chain; and (ii) It prevents premature entry of reactive substrates into the KS active site. To test these hypotheses, we will better define the turnstile mechanism through a comparative study of a normal and a stuttering module. We will also construct and reconstitute PKSs harboring engineered modules that either stutter or ones that show other turnstile-related aberrations, and analyze their properties under multiple turnover conditions.
Assembly line polyketide synthases are multifunctional catalytic machines responsible for the biosynthesis of structurally complex and diverse natural products including many antibiotics. The objective of this research is to advance a fundamental understanding of polyketide biosynthetic mechanisms on an assembly line, and to parlay this knowledge into practical methods for the discovery and engineering of new antibiotics. The central model of interest to us is the polyketide synthase responsible for the biosynthesis of the widely used antibiotic, erythromycin.
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