Browsing by Subject "molybdenum"

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    Kinetic consequences of the endogenous ligand to molybdenum in the DMSO reductase family: a case study with periplasmic nitrate reductase
    (Springer, 2021-02) Mintmier, Breeanna; McGarry, Jennifer M.; Bain, Daniel J.; Basu, Partha; Chemistry and Chemical Biology, School of Science
    The molybdopterin enzyme family catalyzes a variety of substrates and plays a critical role in the cycling of carbon, nitrogen, arsenic, and selenium. The dimethyl sulfoxide reductase (DMSOR) subfamily is the most diverse family of molybdopterin enzymes and the members of this family catalyze a myriad of reactions that are important in microbial life processes. Enzymes in the DMSOR 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. Molybdenum coordination has long been proposed to impact the catalytic activity of the enzyme. Specifically, the molybdenum-coordinating residue may tune substrate preference. As such, molybdopterin enzyme periplasmic nitrate reductase (Nap) is utilized as a vehicle to understand the substrate preference and delineate the kinetic underpinning of the differences imposed by exchanging the molybdenum ligands. 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 properties of these variants are discussed and compared with those of the native enzyme, providing quantitative information to understand the function of the molybdenum-coordinating residue.
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    Molecular cloning, heterologous expression, and steady-state kinetics of camplyobacter jejuni periplasmic nitrate reductase
    (2020-08) Mintmier, Breeanna; Basu, Partha; Georgiadis, Millie; Deiss, Frédérique; Minto, Robert
    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 (NO3-) to nitrite (NO2-), 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.