The Pseudomonas aeruginosa PAO1 metallo flavoprotein D-2-hydroxyglutarate dehydrogenase requires Zn2+for substrate orientation and activation

Pseudomonas aeruginosa PAO1 D-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital L-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes D-malate as an alternative sub-strate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to ratio-nalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or D-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with D-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover.

Automated summary

Researchers have found that zinc ion (Zn2+) plays a crucial role in the catalytic process of Pseudomonas aeruginosa PAO1 D-2-hydroxyglutarate dehydrogenase (PaD2HGDH). It activates the water molecule coordinated to the metal, leading to the correct orientation and activation of the substrate in the enzyme's active site. This finding is important for understanding the catalytic mechanism of PaD2HGDH and other metalloflavoenzymes.