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MOLECULAR CLONING, HETEROLOGOUS EXPRESSION, AND STEADY-STATE KINETICS OF CAMPYLOBACTER JEJUNI PERIPLASMIC NITRATE REDUCTASE

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posted on 29.06.2020, 18:48 by Breeanna Nicole Mintmier
Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate to nitrite, and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.

History

Degree Type

Doctor of Philosophy

Department

Chemistry

Campus location

Indianapolis

Advisor/Supervisor/Committee Chair

Dr. Partha Basu

Additional Committee Member 2

Dr. Millie Georgiadis

Additional Committee Member 3

Dr. Frédérique Deiss

Additional Committee Member 4

Dr. Robert Minto

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