Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Keturah Wallace - Johnson C. Smith University
Co-Author(s): Jihye Jung and Bernd Nidetzky, Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
Nitrile reductase from Escherichia coli (EcNR) catalyzes the reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl -7-deazaguanine (preQ1) by using two molecules of nicotinamide adenine dinucleotide phosphate (NADPH). EcNR is a potential biocatalyst for nitrile reduction to amine as well as a potential antimicrobial drug target. The knowledge of the enzymatic mechanism is beneficial not only to develop the enzyme that fits commercial use but also to design enzyme inhibitors. Biochemical and kinetic studies of wild-type enzyme showed that two hydride transfers from NADPH to preQ0 are Rstereospecific and rate-limiting steps in the enzymatic mechanism of EcNR. However, the function of the amino acids in the active site has not been fully understood yet. In order to understand the role of amino acids in the active site, we present primary kinetic isotope effect (KIE) studies with mutant enzyme. Since a (4R)-deuterated NADPH ((4R)-[2H]NADPH) is not commercially available, (4R)-[2H]NADPH was produced by the reduction of NADP+ using alcohol dehydrogenase from Thermoanaerobium brockii in the presence of 2-propanol-d8. The produced coenzyme was confirmed by Proton Nuclear Magnetic Resonance (1H-NMR) and High Performance Liquid Chromatography (HPLC) (purity > 90 %). Kinetic isotope effect on turnover number (Dkcat) was investigated by the enzymatic reduction of preQ0 in the presence of the mutant enzyme with NADPH or 4R-[2H]NADPH. This work is valuable to comprehend functional amino acids in the active site, extending the knowledge about the biocatalytic mechanism of EcNR.
Funder Acknowledgement(s): Jihye Jung, Bernd Nidetzky
Faculty Advisor: Bernd Nidetzky,