Kinetic analysis and engineering of acyltransferase domains from modular polyketide synthases [electronic resource]

ABSTRACT Microorganisms are an invaluable resource in the discovery and development of therapeutically active small molecules. Microorganism-derived natural products became more prevalent following the discovery of penicillin, and by the 1990s nearly 80% of approved drugs were either natural products or natural product analogs. A subsequent decline in efforts to isolate compounds from natural sources by the pharmaceutical industry coincided with the onset of an antibiotic "discovery void, " a period of over two decades during which the discovery of new classes of antibiotics declined sharply. The United States Centers for Disease Control and the World Health Organization have both recently released reports outlining the dangers of not only stalls in antibiotic discovery, but also of the emergence of drug-resistant bacteria. Further, drug-resistant fungi and other microbes, as well as chemotherapy-resistant cancer cell lines push the problem beyond just antibiotics. Society is clearly in dire need of new therapeutics and more specifically, new ways to identify, isolate, and engineer medicinally-relevant compounds. The polyketide class of natural products has seen huge success in the commercial drug arena, with a "hit rate" that is several orders of magnitude above synthetic compound libraries. Polyketides, named for the adjacent methylene and carbonyl functional groups that are characteristic of their structure, act as a wide range of therapeutics. Many polyketides are produced by large, modular enzymatic complexes called polyketide synthases (PKSs), which build polyketides from simple precursors in an assembly line manner. Each module of a PKS is composed of several enzymatic domains, and each domain plays a distinct role in extending and chemically modifying the growing polyketide chain. The acyltransferase (AT) domain is responsible for selection and incorporation of simple Coenzyme A- (CoA-) linked substrates, which are transferred to the phosphopantetheine arm of an adjacent acyl carrier protein (ACP). Due to their role as gatekeepers to building block incorporation, AT domains are commonly targeted in the engineering of PKS assembly lines for the production of novel therapeutic polyketides. Broadly, this dissertation focuses on the characterization and interrogation of the substrate specificity and catalytic mechanism of AT domains. These studies are necessary for the successful engineering of the substrate selection of assembly line PKSs. Further, because PKSs are among the most complex enzyme systems existing in nature, the contributions of these studies to problems of larger enzymological importance cannot be discounted. The understanding of biological machines, especially those involved in secondary metabolism, is considered one of the most important problems facing enzymologists and biochemists today. More specifically, this dissertation begins with a survey of the current state of AT domain engineering efforts—our knowledge of the sequence, structural, and catalytic characteristics of this enzyme; an examination of previous attempts to engineer AT domain substrate specificity; and the best approaches moving forward based on the aforementioned data. We then present the first use of a steady state, continuous assay to characterize the substrate specificity and mechanism of a representative assembly line AT domain from the erythromycin synthase. Because the specificity of an AT domain depends on both interactions with the CoA-linked substrate and the protein-protein interactions necessary for loading of the ACP, we examine the effects of titrating both substrates on AT catalysis. We also examine previously identified substrate specificity-altering mutations, and conclude that most mutations likely degrade specificity for the native substrate rather than enhancing specificity for alternative substrates. While most PKSs contain dedicated AT domains within the assembly line (often called cis-AT domains), some instead utilize stand-alone domains that are said to act in trans. These trans-AT domains have the potential to be important engineering tools, as the activity of an active site knockout cis-AT can be complemented with a trans-AT of differing specificity. We next examine the substrate specificity and catalytic mechanism of several different cis- and trans-AT domains. Differences in the catalytic mechanism between AT configurations has important implications for the substrate specificity engineering of these domains. We also test the generality of our results by complementing the activity of an AT domain knockout in the full erythromycin PKS assembly line in vitro. Finally, we present our progress toward the rational engineering of AT domains. We utilize sequence- and structure-based techniques including conservation analysis, statistical coupling analysis, and crystal structure alignments. Experimental interrogation of the activity of AT site-directed mutants complements this in silico data. Careful analysis of the activity of AT mutants reveals the possible roles of specific residues in determining the nuanced structural and chemical properties necessary for successful catalysis. In summary, this dissertation presents an in-depth analysis of AT domain catalysis and specificity. This work pushes the field closer to the rational engineering of PKS assembly lines for the production of novel polyketide therapeutics, and provides fundamental insight into the inner workings of an extremely complex and amazing molecular machine.

Keywords: Chemical Engineering.
Advisor: Khosla Chaitan
Cochran Jennifer R. advisor. Thesis advisor
Sattely Elizabeth advisor. Thesis advisor