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Specific reduction of a 1‐Cys Prx from Pseudomonas aeruginosa by ascorbate
Author(s) -
Aleixo Silva Rogério,
Domingos Renato,
Netto Luis
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.05010
Subject(s) - pseudomonas aeruginosa , chemistry , cysteine , virulence , biochemistry , hydrogen peroxide , peroxiredoxin , microbiology and biotechnology , superoxide , oxidative phosphorylation , oxidative stress , reactive oxygen species , peroxynitrite , peroxidase , stereochemistry , enzyme , biology , bacteria , gene , genetics
Pseudomonas aeruginosa is a ubiquitous gammaproteobacteria that is the major cause of hospitalar infections of pneumonia and is the major pathogen associated with lung infections in cystic fibrosis patients. Among several defense systems against pathogens, host cells release oxidants, such as: superoxide (O 2 ˙ ‐ ), hydrogen peroxide (H 2 O 2 ) and peroxynitrite during phagocytosis. To overcome this oxidative insult, P. aeruginosa possesses a myriad of strategies, including Cys‐based peroxidases called peroxiredoxins. One of the peroxiredoxins from P. aeruginosa is LsfA that belongs to the Prx6 subgroup, containing only one cysteine and displays the so‐called 1‐Cys Prx mechanism. In addition, LsfA was previously related with P. aeruginosa virulence. Therefore, the aim of the present work is to characterize LsfA in biochemical and structural grounds. Here, we describe two crystallographic structures of LsfA in distinct oxidative states: reduced (Cys‐S ‐ ) and sulfonic acid (Cys‐SO 3 H) at 2.6 and 2.0 Ǻ resolution, respectively. The native dimeric state was confirmed by crystallography and SAXS (Small angle X ray scattering) in the reduced (Cys‐S ‐ ), oxidized (Cys‐SOH) and hyperoxidized (Cys‐SO 2/3 H) states. The LsfA active site topology is slightly different than the human Prx6 homologue, suggesting that these enzymes might present distinct substrate specificities. Furthermore, other differences were observed, such as the orientation of His37 that changes in a redox dependent manner in human Prx6, but not in LsfA. We also investigated the substrates specificities of LsfA. Previously, we observed that LsfA decomposes distinct peroxides at extremely high rates ( k = 10 6 ‐ 10 7 M ‐1 .s ‐1 ). However, a reductant is required to turnover LsfA, but the identity of this compound is still unknown to LsfA. Therefore, we investigated if thioredoxin or glutathione/glutaredoxin systems from P. aeruginosa could act as LsfA reductants. We employed recombinant enzymes and coupled assays and we observed that the thioredoxin or glutathione/glutaredoxin systems failed to support the peroxidase activity of LsfA. Previously, we observed that ascorbate reduces the sulfenic acid form of LsfA with a second order constant rate of 10 3 M ‐1 .s ‐1 , using a competition assay based on DCPIP (2,6–dichlorophenolindophenol). Therefore, we investigated the effects of ascorbate on P. aeruginosa growth, employing a bacterial strain with the LsfA gene deleted. Wild type and ΔlsfA strains did not grew on ascorbate as a sole carbon source. Furthermore, no ascorbate toxicity up to 300 mM concentration was observed, when growing both strains in a minimum media with glucose as carbon source. Studies are underway to understand how P. aeruginosa cells uptake and export ascorbate. Furthermore, the dependencies of P. aeruginosa cells on LsfA and ascorbate to decompose exogenously added peroxides will be investigated.