Dataset Information

Uncovering Zn²⁺ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

ABSTRACT: 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 Zn²⁺, Co²⁺, or Mn²⁺ 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 Zn²⁺, the Zn²⁺:protein stoichiometry was 2:1, yielding an enzyme with ∼40 s^-1k_cat for d-malate. Treatment with 1 mM EDTA decreased the Zn²⁺: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 Zn²⁺ is essential for PaD2HGDH activity. The presence of Zn²⁺ increased the flavin N³ atom pK_a value to 11.9, decreased the flavin ε₄₅₀ 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 Zn²⁺-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn²⁺, Co²⁺, Cd²⁺, Mn²⁺, or Ni²⁺ 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 Zn²⁺-dependent metallo flavoprotein, which requires Zn²⁺ as an essential cofactor for enzyme activity.

SUBMITTER: Quaye JA

PROVIDER: S-EPMC10025160 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Uncovering Zn2+ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

Quaye Joanna A JA Gadda Giovanni G

The Journal of biological chemistry 20230211 3

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 Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator ...[more]

PMID: 36775126

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Project description:The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

Dataset Information

Uncovering Zn²⁺ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

Publications

Uncovering Zn<sup>2+</sup> as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

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