Uncovering Zn as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

Pseudomonas aeruginosa couples the oxidation of d-2-hydroxyglutarate (D2HG) to l-serine biosynthesis for survival, using d-2-hydroxyglutarate dehydrogenase from P. aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn, Co, or Mn but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or d-malate. Upon purifying PaD2HGDH with 1 mM Zn, the Zn:protein stoichiometry was 2:1, yielding an enzyme with ∼40 sk for d-malate. Treatment with 1 mM EDTA decreased the Zn:protein ratio to 1:1 without changing the kinetic parameters with d-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100 mM EDTA treatment, suggesting that Zn is essential for PaD2HGDH activity. The presence of Zn increased the flavin N atom pK value to 11.9, decreased the flavin ε at pH 7.4 from 13.5 to 11.8 mM cm, and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn, Co, Cd, Mn, or Ni to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn-dependent metallo flavoprotein, which requires Zn as an essential cofactor for enzyme activity.