Uncovering Zn2+ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.103007
ISSN1083-351X
AutoresJoanna Afokai Quaye, Giovanni Gadda,
Tópico(s)Enzyme Structure and Function
ResumoPseudomonas 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−1 kcat 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 pKa 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. 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−1 kcat 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 pKa 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. Metals are an essential class of elements necessary for the survival of all life forms, including bacteria, plants, and humans (1Jennette K.W. The role of metals in carcinogenesis: biochemistry and metabolism.Environ. Health Perspect. 1981; 40: 233-252Crossref PubMed Google Scholar, 2Pokorska-Niewiada K. Rajkowska-Myśliwiec M. 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Despite the suggestion of PaD2HGDH as a metallo flavoprotein, there is no understanding of its metal composition, binding and coordination, significance and importance, and activation mechanism. In this study, His-tagged PaD2HGDH from P. aeruginosa PAO1 has been recombinantly expressed, purified to high levels, and investigated for its activity in the presence of Zn2+. Zn2+ has been uncovered as an essential cofactor for PaD2HGDH, rather than an enzyme activator, identifying the enzyme as a metallo flavoprotein. The kinetic and inductively coupled plasma-mass spectrometry (ICP-MS) analysis data of PaD2HGDH treated with and without EDTA are discussed. This study proposes a mechanism for metal reactivation of inactive PaD2HGDH with potential protein ligands for metal binding in PaD2HGDH. To characterize PaD2HGDH as purified in its kinetic properties, the reductive-half reaction of the enzyme purified in the absence of ZnCl2 (vide infra) in the purification buffers was investigated by following the substrate-induced absorbance changes of the enzyme-bound flavin at 450 nm. Time-resolved absorption spectroscopy at varying concentrations of d-malate showed incomplete flavin reduction, that is, ∼15%, in two reaction phases (Fig. 1) and irrespective of the substrate concentration used. Similar results were obtained with D2HG (Fig. 1). These data are consistent with the data previously reported for the human enzyme, for which no explanation was provided to account for the partial flavin reduction (51Toplak M. Brunner J. Schmidt J. Macheroux P. Biochemical characterization of human D-2-hydroxyglutarate dehydrogenase and two disease related variants reveals the molecular cause of D-2-hydroxyglutaric aciduria.Biochim. Biophys. 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