Structural studies of enzymes involved in antibiotic resistance and phosphonate degradation

Antibiotic;Phosphonate;X-ray crystallography;enzymology

Agarwal, Vinayak

关键词: Antibiotic;    Phosphonate;    X-ray crystallography;    enzymology;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/42440/Vinayak_Agarwal.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Biological chemistry revolves around the abiogenetic interplay of high energy labile chemical linkages which shuttle energy and chemical potential, and stable and degradation resistant bonds that make up the biological polymers which offer structural and functional support. Insights into secondary metabolism in microorganisms have shed light on fascinating arrays of genetically encoded small molecule natural products which have utilized the chemical frameworks of transient high energy compounds, and replaced key labile bonds with those borrowed from stable structural molecules. Compounds such as these can be designed as mimics of enzymatic transition states, which thus inhibit the respective enzymes by binding to them, but are not turned over to products.One such example is the Trojan horse tRNA synthetase inhibitor antibiotic microcin C7, which mimics the aminoacyl adenylate intermediate of the tRNA synthetase enzyme by replacing the high energy phosphodiester bond of the enzyme intermediate with a stable phosphoramine-amide linkage. Second examples are the phosphonate molecules which replace the high energy phosphoryl and carboxyl ester moieties of various enzyme intermediates by stable phosphonate linkages. These compounds in addition to possessing bioinhibitory activities are also thought to be store houses of carbon and phosphorous in the microbial metabolome as they cannot be easily degraded. The body of work presented in this dissertation delves into molecular mechanisms of enzymatic degradation of the stable phosphoramine-amide bond of microcin C7, as well as the hydrolysis of the phosphonate bond in a ubiquitous phosphonate molecule- phosphonoacetate. Both these studies, presented in the form of two chapters, involve the atomic resolution structure determination of the enzyme catalysts during various stages of their catalytic cycles, and in complex with different substrate molecules which delineate their specificity and selectivity. All structural investigations have been followed by extensive biochemical validation of the structural results. These results have allowed us to postulate the reaction mechanisms for these catalysts. Our proposals reveal how enzymatic architectures have been borrowed from primary metabolism, and the enzyme active sites slightly tweaked to achieve remarkable and unprecedented chemical transformations by these enzymes of microbial secondary metabolism. Our studies also have implications for the design of inhibitors of these enzymes, as similar mechanisms may be used to avert the bioinhibitory action of various antibiotics by drug resistant pathogenic microorganisms.