Uncovering Zn(2+) 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(2+), Co(2+), or Mn(2+) 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(2+), the Zn(2+):protein stoichiometry was 2:1, yielding an enzyme with ∼40 s(−1)k(cat) for d-malate. Treatment with 1 mM EDTA decreased the Zn(2+):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(2+) is essential for PaD2HGDH activity. The presence of Zn(2+) increased the flavin N(3) atom pK(a) value to 11.9, decreased the flavin ε(450) at pH 7.4 from 13.5 to 11.8 mM(−1) cm(−1), and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn(2+)-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn(2+), Co(2+), Cd(2+), Mn(2+), or Ni(2+) 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(2+)-dependent metallo flavoprotein, which requires Zn(2+) as an essential cofactor for enzyme activity.