Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

Cell-to-cell communication by bacteria is essential for the regulation of gene expression important in colonization, biofilm formation, virulence and other processes. This communication is called "quorum-sensing" and is mediated by small molecules called autoinducers. One major class of autoinducers used by gram-negative bacteria is N-acyl homoserine lactones (AHL's). Enzymes capable of disrupting this communication are called "quorum-quenching" catalysts and have proven to be invaluable biochemical tools for understanding quorum-sensing pathways. Quorum-quenching enzymes hold promise for application in anti-biofouling, agriculture, aquaculture, bioremediation and other synthetic biology settings. However, the mechanisms that these enzymes use to recognize and process their substrates are not well characterized. A better understanding of selectivity and catalysis would enable the production of variant enzymes to recognize specific quorum-sensing signals to serve as catalysts for various applications.

Aminotransferases are a class of pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze a myriad of biochemical reactions involved in several metabolic processes. The importance of these enzymes is further underscored because some of them have been identified as drug targets. For example, inhibition of γ-aminobutyric acid aminotransferase (GABA-AT) was proven effective in the treatment of many neurological disorders, and inhibition of ornithine aminotransferase (OAT) was shown to suppress the growth of hepatocellular carcinoma (HCC). Potent mechanism-based inactivators can be rationally designed against these PLP-dependent drug targets, but one of the remaining challenges is the lack of selectivity against other PLP-dependent off-target enzymes such as aspartate aminotransferase. A better understanding of enzyme selectivity and mechanism would enable the design of molecules capable of displaying desirable specificity of inactivation discerning drug-targets and off-targets.

The goal of this dissertation is to provide better understanding of selectivity and catalysis in quorum-quenching enzymes, and selectivity and mechanisms of PLP-dependent aminotransferases. The structural and biochemical characterization of AidC, an N-acyl homoserine lactone lactonase, was carried out using X-ray crystallography and other biochemical techniques. The selectivity of a mechanism-based inactivator was tested in a PLP-dependent aminotransferase drug-target (OAT) and off-target (Asp-AT) using X-ray crystallography and enzyme assays. The proposed mechanism was confirmed by high-resolution mass spectrometry.

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