Date of Award
Doctor of Philosophy (PhD)
Flavin-dependent enzymes are characterized by an amazing chemical versatility and play important roles in different cellular pathways. The FAD-containing choline oxidase from Arthrobacter globiformis oxidizes choline to glycine betaine and retains the intermediate betaine aldehyde in the active site. The reduced FAD is oxidized by oxygen. Glycine betaine is an important osmoprotectant accumulated by bacteria, plants, and animals in response to stress conditions. The FMN-containing nitronate monooxygenase detoxifies the deadly toxin propionate 3-nitronate which is produced by plants and fungi as defense mechanism against herbivores. The catalytic mechanism of fungal nitronate monooxygenase (NMO) was characterized, but little is known about bacterial NMOs.
In this dissertation the crystal structure of choline oxidase in complex with glycine betaine was solved and the roles of the residue F357 in the oxidative half reaction investigated by combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects and molecular dynamics simulations. Expression trials of human choline dehydrogenase were carried out and a homology model based on choline oxidase was generated. A bacterial nitronate monooxygenase from Pseudomonas aeruginosa PAO1 Pa-NMO was kinetically and structurally characterized and four conserved motifs were described that identify Class 1 NMO. Two hypothetical NMOs from P. aeruginosa PAO1 and one from Helicobacter pylori not carrying the motifs of Class 1 NMO were cloned and tested for nitronate monooxygenase activity.
The crystal structure of choline oxidase in complex with glycine betaine highlighted two different conformations of loop 250-255 at the dimer interface that is proposed to control substrate access to the active site. The side chain of F357 was associated with a slow isomerization in the oxidation of the reduced FAD of choline oxidase. The first crystal structure of an NMO highlighted active site residues for site-directed mutagenesis studies. The gene function prediction for NMO was improved with the four conserved motifs of Pa-NMO. The two hypothetical NMOs from P. aeruginosa PAO1 were shown to possess a different enzymatic activity and to identify two distinct classes of enzymes. The hypothetical NMO from H. pilory did not exhibit NMO activity and contained iron as cofactor.
Salvi, Francesca, "Kinetic and Structural Studies on Flavin-dependent Enzymes involved in Glycine Betaine Biosynthesis and Propionate 3-nitronate Detoxification." Dissertation, Georgia State University, 2015.