Date of Award
Master of Science (MS)
NADH:quinone oxidoreductase (NQO) from Pseudomonas aeruginosa PA01 is an FMN-dependent enzyme that utilizes NADH to catalyze the two-electron reduction of a wide variety of benzoquinones and naphthoquinones. The two-electron quinone reduction by NQO is thought to play a detoxifying role in P. aeruginosa PA01 by avoiding the formation of semiquinone radicals known to cause oxidative stress in cells. Crystal structures of NQO demonstrate the enzyme contains two domains: a TIM-barrel domain and an extended domain which are connected through two βα loops. The TIM-barrel domain is the most common enzyme fold found in nature and is often utilized as a target to engineer catalytic functions during the de novo synthesis of enzymes. NQO is a mostly unexplored enzyme at the time of this thesis, where only its mechanistic and structural properties have been elucidated. In this thesis, the functional roles of two non-catalytic residues that form the active site of NQO are investigated by employing UV-visible absorption spectroscopy, molecular dynamics, and steady-state kinetics. The thesis presented below highlights the importance of non-catalytic residues in modulating the structural, biophysical, and kinetic properties of NQO, which should be taken into account during the de novo synthesis of TIM-barrel containing enzymes.
Previously solved crystal structures of NQO demonstrated the βα loop 3 (residues 75-86) from the TIM-barrel domain samples an open conformation in the ligand-free form and a closed conformation in the ligand-bound form. The relationship between loop 3 rigidity and turnover rates in NQO was investigated by replacing the conserved P78 with a glycine. Circular dichroism, fluorescence spectroscopy, and UV-visible absorption spectroscopy were employed to demonstrate the P78G mutation minimally altered the secondary structure elements of the protein and the active site environment surrounding the flavin cofactor when compared to wild-type NQO. The gate in NQO was determined to consist of loop 3 and the extended domain, where molecular dynamics demonstrated the substrate-free form of NQO samples more open gate conformations following the P78G mutation. The steady-state kinetic parameters of the mutant and wild-type enzymes revealed the mutation led to a minimal increase in the kcat/KCoQ0 value, suggesting the mutation may promote the rate of association with the quinone substrate. The results presented in this investigation suggest the structural rigidity of loop 3 plays a role in modulating domain-domain interactions that may increase the rate of substrate association in NQO.
In the second part of this thesis, the relationship between the protonation state of Y277 and the flavin cofactor’s absorption spectrum was investigated in NQO. Y277 is mainly exposed to the solvent in the active site of NQO, with one exception being a 3.0 Å separation between the oxygen atom of Y277 and the C7 methyl group of the flavin cofactor. The UV-visible absorption spectrum of NQO was investigated as a function of pH and compared to those of a mutant enzyme NQO-Y277F, which indicated Y277 deprotonates at high pH. A combination of UV-visible absorption spectroscopy and QM/MM computations were utilized to determine the effect of Y277 deprotonation on flavin’s absorption peak intensities and wavelengths. Both the biochemical experiments and computational calculations suggest that in the absence of solution ions, deprotonating Y277 will significantly alter the absorption spectrum of flavin; however, in the presence of added Na+ and Cl- ions, no spectral changes are observed between protonated and unprotonated Y277. QM/MM simulations of NQO with Y277 in the neutral and anionic forms revealed either Na+ ions move closer to the protein surface and/or Cl- ions move away from the surface as Y277 deprotonates in the active site. The results presented in this portion of the thesis establish that the solution ions surrounding NQO can interact with the negative point charge at residue 277 through a long-distance counterion effect to prevent the flavin cofactor’s absorption spectrum from being altered.
Dratch, Benjamin and Gadda, Gioavanni, "Functional Importance of Non-Catalytic Residues in the Active Site of NADH:Quinone Oxidoreductase From Pseudomonas aeruginosa PA01." Thesis, Georgia State University, 2021.
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