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Biophysical and Kinetic Investigation of Metallo-Flavoenzyme D-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1
Summary
Scientists studied an enzyme that helps the bacterium Pseudomonas aeruginosa (a common cause of infections, especially in hospitals) survive, hoping to find weaknesses that could lead to new antibiotics. Interestingly, they found that tiny plastic particles from polystyrene and polyethylene, common plastics found in everyday products, can block this enzyme's activity, suggesting that nanoplastic pollution might interfere with bacterial function in ways we don't fully understand yet. While this doesn't directly tell us about human health effects, it's a reminder that the growing amount of microplastics in our environ
D-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH) is a Zn2+- and FAD-dependent metallo-flavoenzyme that catalyzes the oxidation of D-2-hydroxyglutarate to 2-ketoglutarate in bacterial metabolism. Since this enzyme participates in a metabolically important pathway in P. aeruginosa, defining the molecular features that control its activity is important for understanding flavoenzyme catalysis and for identifying new opportunities for antibacterial targeting. This dissertation presents a biophysical and kinetic investigation of PaD2HGDH, with emphasis on how the metal cofactor, active-site electrostatics, and environmental perturbations regulate catalysis. Spectroscopic and biochemical analyses showed that replacement of the native Zn2+ with other divalent metal ions perturbs the flavin environment, alters the ionization behavior of the enzyme-bound cofactor, and changes catalytic activity without major disruption of the overall protein fold. Kinetic studies further established that the metal cofactor does not affect turnover in a simple uniform manner, but instead modulates specific steps along the catalytic pathway. Substitution with Ni2+ increased the contribution of substrate dissociation and product release to catalysis, whereas substitution with Cd2+ decreased substrate capture efficiency and slowed flavin reduction. These results show that the metal ion fine-tunes the balance among substrate binding, flavin chemistry, and conformational motions during turnover. This dissertation also defines the mechanistic role of the conserved residue K339 near the active site. Site-directed mutagenesis combined with rapid-reaction kinetics showed that the positive charge at this position promotes substrate binding, contributes to proper substrate orientation for hydride transfer, and helps organize the ionization and conformational equilibria required for efficient catalysis. Comparative analysis of D2HGDH homologs further places PaD2HGDH in a broader structural and mechanistic context. Finally, nanoplastic studies showed that polystyrene and polyethylene inhibit PaD2HGDH through distinct noncompetitive surface interactions, with polyethylene acting as the more potent perturbant and reshaping the conformational landscape of catalysis. Overall, the results establish that PaD2HGDH activity depends on a finely tuned interplay among metal coordination, flavin reactivity, electrostatics, protein dynamics, and environmental context.