Enabling a rapid transition from synthase to structure

Project Summary. Polyketide natural products have played, and will continue to play, critical roles in cancer
chemotherapy. Though they are potent and often tumor-selective agents, access to gram scale supplies of these
agents complicates their clinical translation. Current solutions, including chemical synthesis and strain
optimization, have provided modest success, but our lack in understanding the machines responsible for their
production ultimately limits the ability to attain effective levels of production. Type I polyketide synthases (PKSs)
are large multifunctional enzymes that are organized into modules of discrete enzymatic proteins, minimally
containing a β-ketoacyl synthase, an acyltransferase, and an acyl carrier protein (ACP). While an ideal size for
cryo-electron microscopy (cryo-EM), only a few Type I PKS structures currently exist, and of these many lack
resolution of their ACP domain. Like many large proteins, they exist naturally in a highly dynamic state, wherein
their activity operates through discrete, mechanical processes. In most Type I PKSs, biosynthesis arises through
assembly of ketide units, which are shuttled between the enzymatic domains by means of the ACP. Here, the
complex movement between domains in each module results in synchronic capture of multiple states of the
same protein, therein complicating structural evaluation. In this program, our team explores the pathways to
enable the selective evaluation of state trapped Type I PKSs. Through two specific aims, we offer a unique
solution towards isolating PKS megasynthases from host cells or producing them through recombinant
technologies for cryo-EM studies. Overall, two key fundamental discoveries will arise from this program: an
approach to isolating PKS enzymes for cryo-EM studies by directly challenging the current methods; and a
primary understanding of the selectivity and mechanics involved in PKS catalysis. We anticipate that this
program will offer not only a basis for structural resolution of large PKS enzymes, but also offer insight into
modular and intermodular dynamics within these complex multimodular synthases.

Grant Number: 1R21CA280558-01A1
NIH Institute/Center: NIH
Principal Investigator: Michael Burkart