Abstract Summary/Description
Pseudomonas aeruginosa PAO1 is a gram-negative bacterium responsible for ~10% of all nosocomial infections in the U.S. In 2017 alone, P. aeruginosa infections caused ~3000 deaths and ~30,000 nosocomial infections.1 P. aeruginosa’s survival relies on the ability of D-2-hydroxyglutarate dehydrogenase (PaD2HGDH) to reproduce 2-ketoglutarate from D-2-hydroxyglutarate during L-serine biosynthesis.2-5 Since the knockout of the PaD2HGDH gene impairs P. aeruginosa growth, PaD2HGDH is a potential therapeutic target against P. aeruginosa.2 PaD2HGDH has been established as a Zn2+ and FAD-dependent dehydrogenase that uses Zn2+ to orient and polarize its substrate for catalysis.4,5 In closely related flavin-dependent enzymes such as the FMN-dependent -hydroxy acid oxidizing enzymes and the glucose-methanol-choline (GMC) class of enzymes, a catalytic base, usually a histidine residue, is responsible for activating the substrate for catalysis.6,7 Amino acid sequence alignment of PaD2HGDH with other FAD-dependent -hydroxy acid oxidizing enzymes revealed a fully conserved H421 residue3, whose active site location is topologically conserved for catalytic bases in most GMC-type enzymes.6 Additionally, a recent mechanistic study on PaD2HGDH revealed the requirement of a proton acceptor for enzyme catalysis after the alcohol proton displacement by the Zn2+ cofactor.7 However, the role of H421 in PaD2HGDH catalysis has not been established. In this study, site-directed mutagenesis was used to replace H421 with glutamine, asparagine, phenylalanine, or cysteine. The variant enzymes were purified in the presence of 1 mM ZnCl2 and investigated for their mechanistic, biophysical, and structural properties. From UV-visible absorption spectroscopy and ICP-MS data, both the FAD and Zn2+ cofactors are bound to all the variant enzymes. Circular dichroism spectroscopy revealed a similar overall fold, with comparable secondary and tertiary structural elements across all enzymes. Additionally, there was an observed 400-fold decrease in the variant enzymes’ activities, with a ~70% decrease in flavin reduction in comparison to the wild-type enzyme. The data suggest that the fully conserved H421 residue is important for enzyme catalysis and is likely the proton acceptor required for flavin reduction in PaD2HGDH. References 1. Center for Disease Control and Prevention (2019), Antibiotic Resistance Threats in the United States, U.S .Department of Health and Human Services, CDC, Atlanta, GA:1-114 2. Guo, X. et al. (2018) d-2-Hydroxyglutarate dehydrogenase plays a dual role in l-serine biosynthesis and d-malate utilization in the bacterium Pseudomonas stutzeri. J Biol Chem. 293(40):15513-15523 3. Quaye, J.A., Gadda G. (2020) Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1. Biochemistry. 59(51):4833-4844 4. Quaye, J.A., Gadda G. (2023) Uncovering Zn2+ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase. J Biol Chem. 299(3):103007 5. Quaye, J.A., Gadda G. (2023) The Pseudomonas aeruginosa PAO1 metallo flavoprotein d-2-hydroxyglutarate dehydrogenase requires Zn2+ for substrate orientation and activation. J Biol Chem. 299(3):103008 6. Romero, E., Gadda G. (2014) Alcohol oxidation by flavoenzymes. Biomol Concepts. (4):299-318 7. Quaye, J.A., Gadda G. (2024) Metal-Triggered FAD Reduction in D 2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1. ACS Bio & Med Chem Au. doi: 10.1021/acsbiomedchemau.4c00108
