of the parent R01 grant is included here: 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. 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. 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. 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. 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. The proposed supplement activities do not change the goals or approaches of the parent project. The supplement includes both research activities and training activities. Research activities will focus on Aim 3 above, with a focus on in-depth comparative analysis of the prototypical (non-stuttering) Module 1 of the 6- deoxyerythronolide B synthase and the atypical (stuttering) Module 5 of the NOCAP synthase. Training activities will prepare Ms. Guzman enhance her knowledge of enzyme chemistry and engineering as well as her research skills in this area. Additional training activities include a tailor-made program to help her improve her written and oral communication skills, given Ms. Guzman?s goal of pursuing an academic career.
The parent project aims to study assembly line polyketide synthases that make structurally complex and diverse natural products including many antibiotics. Although the proposed supplement activities do not change the goals of the parent project, they will enhance our understanding of vectorial polyketide biosynthesis. They will also boost the participation of an under-represented minority doctoral student.
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