Date of Award
Doctor of Philosophy (PhD)
Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is an FAD-dependent enzyme that catalyzes the oxidative deamination of D-arginine to generate the corresponding α-keto acid and ammonia. The enzyme is similar to D-amino acid oxidase, except that PaDADH is a strict dehydrogenase with no oxygen reactivity. The enzyme exhibits broad substrate specificity and can oxidize 17 of the 20 common amino acids. The best substrates for the enzyme are D-arginine and D-lysine based on the second order rate constant kcat/Km values.
The 3D structure of the DADH-iminoarginine complex suggests that E87 engages in an electrostatic interaction with the positively charged guanidinium group of D-arginine. The 3D structure also displays a hydrogen bonding network of water molecules that connects H48 to the substrate α-amine, suggesting it may serve as catalytic base. E87 and H48 were mutated to produce E87L and H48F variants, and pH effect kinetic approaches with zwitterionic and cationic substrates were employed. The data, in combination with previous results on Y53 and Y249 variants, suggests that there is no catalytic base, since the catalytic pKa is present in all variants. The results are also consistent with E87 required to be deprotonated to bind cationic substrates.
In the second part of this dissertation, a presumed nitronate monooxygenase from P. aeruginosa, PA1024, is characterized. PA1024 is determined to not possess NMO activity and instead catalyze the two-electron reduction of quinones via the oxidation of NADH. The enzyme has a strict preference for NADH over NADPH. The functional annotation and bioinformatics identifies a novel class of FMN-dependent NADH:quinone oxidoreductases (NQO) with a TIM-barrel fold. The pH effects reveal that PA1024 has two regimes of activity at low and high pH dictated by the deprotonation of an enzymatic residue with pKa ~7. In addition, the 3D X-ray structure of PA1024 in complex with the reaction product NAD+ is solved. The structure reveals the structural basis for the strict NADH specificity, which relies on a steric constraint imposed by a nearby P78 and Q80. The structure also reveals a conformational gating mechanism of Q80 and an unusual interrupted helix structural pattern to bind the pyrophosphate of NAD+.
Ball, Jacob A., "Mechanistic Studies on D-arginine Dehydrogenase and Functional Annotation of a Novel NADH:quinone Oxidoreductase (PA1024)." Dissertation, Georgia State University, 2018.
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