Our research on polyketide assembly lines is helping bring about a paradigm shift for how sets of component enzymes cooperate to biosynthesize polyketide natural products. The updated definition of a module, with the ketosynthase domain positioned at its downstream end, affects every level of modular polyketide synthase enzymology. Each of these levels must be further explored to achieve our long-term goal of reprogramming polyketide assembly lines to synthesize designer molecules and accelerate the drug discovery process. Our highest-resolution proposal is to study how ketosynthases gatekeep such that only one type of polyketide intermediate is selected by a module to be further elongated by the downstream assembly line (Specific Aim 1). This will be accomplished through methodology we have developed to crystallographically observe polyketides bound in ketosynthase active sites and measure the activity of ketosynthases mutated at suspected gatekeeping residues; engineered triketide synthases will also aid in this effort. From structures determined by our lab and others, we hypothesize that several uncharacterized domain interfaces are present within modules. We seek to structurally elucidate these interfaces within the context of the newly-defined module (Specific Aim 2). Thus, through crystallography and cryo-electron microscopy the structures of multidomain fragments possessing upstream processing enzymes and a downstream ketosynthase will be determined. We will also continue our efforts to characterize transient interfaces that form during the reaction cycle as acyl carrier protein domains present polyketide intermediates to cognate enzymes for catalysis. Our lab has collected several pieces of structural evidence for higher-order architecture. In the bacillaene polyketide synthase, a three-helix element adjacent to the ketosynthase domain seems to zipper homodimeric assembly lines into ~100 MDa assembly sheets observable within Bacillus subtilis cells. We propose to understand the structures of such biosynthetic megacomplexes by reconstituting them in vitro and observing them through electron microscopy (Specific Aim 3). Our lab is already visualizing Pks12 from Mycobacterium tuberculosis both in its ?bimodular? and its polymeric assembly line states. We seek to determine how modules stack to construct higher-order architecture in such systems. Through investigations at each of these levels, an overall picture of the architectures and activities of polyketide assembly lines will emerge that will be particularly significant to the future engineering of these medicinally-relevant molecular machines.

Public Health Relevance

Modular polyketide synthases are enzymatic factories that biosynthesize many of the most important human medicines, such as antibacterials, antifungals, immunosuppressants, and anticancer agents. A recent discovery about how components of these assembly lines co-migrate during evolution has not only revolutionized our understanding of them, but also provided promising new directions to further elucidate their architectures and activities as well as engineer them to produce new molecules and medicines.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Bond, Michelle Rueffer
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Texas Austin
Schools of Arts and Sciences
United States
Zip Code
Liu, Chenguang; Yuan, Meijuan; Xu, Xu et al. (2018) Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase. J Struct Biol 203:135-141
Vander Wood, Drew A; Keatinge-Clay, Adrian T (2018) The modules of trans-acyltransferase assembly lines redefined with a central acyl carrier protein. Proteins 86:664-675
Wagner, Drew T; Zhang, Zhicheng; Meoded, Roy A et al. (2018) Structural and Functional Studies of a Pyran Synthase Domain from a trans-Acyltransferase Assembly Line. ACS Chem Biol 13:975-983
Wagner, Drew T; Zeng, Jia; Bailey, Constance B et al. (2017) Structural and Functional Trends in Dehydrating Bimodules from trans-Acyltransferase Polyketide Synthases. Structure 25:1045-1055.e2
Stevens, D Cole; Wagner, Drew T; Manion, Hannah R et al. (2016) Methyltransferases excised from trans-AT polyketide synthases operate on N-acetylcysteamine-bound substrates. J Antibiot (Tokyo) 69:567-570
Bailey, Constance B; Pasman, Marjolein E; Keatinge-Clay, Adrian T (2016) Substrate structure-activity relationships guide rational engineering of modular polyketide synthase ketoreductases. Chem Commun (Camb) 52:792-5
Keatinge-Clay, Adrian T (2016) Stereocontrol within polyketide assembly lines. Nat Prod Rep 33:141-9
Zeng, Jia; Wagner, Drew T; Zhang, Zhicheng et al. (2016) Portability and Structure of the Four-Helix Bundle Docking Domains of trans-Acyltransferase Modular Polyketide Synthases. ACS Chem Biol 11:2466-74
Gay, Darren C; Wagner, Drew T; Meinke, Jessica L et al. (2016) The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex. J Struct Biol 193:196-205
Xie, Xinqiang; Garg, Ashish; Keatinge-Clay, Adrian T et al. (2016) Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues. Biochemistry 55:1179-86

Showing the most recent 10 out of 27 publications