D-Arginine dehydrogenase (DADH) catalyzes the oxidation of D-arginine to imino arginine using FAD as the cofactor. The enzyme is part of a recently discovered two-enzyme complex from Pseudomonas aeruginosa involved in arginine utilization. Function of the enzyme within the organism is unknown. Work on this enzyme has been undertaken to understand the structure as well as its reaction mechanism so as to eventually assign a function to the enzyme within the physiological context. In the reductive half-reaction 2 e- and 1 H+ are transferred from the amino acid substrate to FAD cofactor. In the oxidative half-reaction the reducing equivalents from the FAD cofactor are passed to an electron acceptor that is yet to be discovered. The enzyme has been established to have no reactivity with O2. Choline oxidase (CHO) from Arthrobacter globiformis is a well characterized member of Glucose-Methanol-Choline Superfamily that reacts with molecular O2. It catalyzes the oxidation of choline to glycine betaine mediated by betaine aldehyde intermediate using FAD as the cofactor and O2 as the oxidant to regenerate oxidized FAD for further reaction. Glycine betaine, the product of the reaction is an important osmolyte that regulates nutrients for plants under stressful conditions. Therefore it is of commercial interest to genetically engineer crops that do not typically possess competent pathways for glycine betaine synthesis.
In this dissertation molecular details concerning the reductive half-eaction of DADH and oxidative half-reaction of CHO have been studied using a combination of steady state kinetics, rapid kinetics, pH, multiple substrates, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects or computational approaches.
In DADH, the oxidation of amino acid substrate by FAD has been shown to most likely proceed via hydride transfer mechanism in the reductive half-reaction with Glu87, Tyr53, Tyr249 and His48 emerging as key players in substrate binding, catalysis or for up keeping the integrity of the FAD cofactor. In CHO, the oxidative half-reaction proceeds without stabilization of any reaction intermediates with H atom from reduced FAD and H+ from solvent or solvent exchangeable site occurring in the same kinetic step.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:scholarworks.gsu.edu:chemistry_diss-1098 |
| Date | 12 August 2014 |
| Creators | Gannavaram, Swathi |
| Publisher | ScholarWorks @ Georgia State University |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Chemistry Dissertations |
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