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. 2015 Apr 27;10(4):e0124427.
doi: 10.1371/journal.pone.0124427. eCollection 2015.

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

Giuliana Donadio  1 , Carmen Sarcinelli  1 , Elio Pizzo  1 , Eugenio Notomista  1 , Alessandro Pezzella  2 , Carlo Di Cristo  3 , Federica De Lise  1 , Alberto Di Donato  1 , Viviana Izzo  4
Affiliations

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

Giuliana Donadio et al. PLoS One. .

Abstract

Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Starting substrates.
(A) Structures of 2-phenylethanol and o-xylene. (B) Substrates used for the ToMO-catalyzed biotransformations presented in this study.
Fig 2
Fig 2. Hydroxylated products.
Starting substrates and corresponding hydroxylated products obtained through ToMO-catalyzed biotransformation of (A) 2-phenoxyethanol and (B) phthalan.
Fig 3
Fig 3. Docking of intermediates in ToMO active site.
Docking of the two possible arenium intermediates (4PT and 5PT) deriving from phthalan in the active site of ToMOA. In the case of 4PT, θ is the torsion angle Fe(2)-O-C4-C9, whereas, in the case of 5PT, θ is the torsion angle Fe(2)-O-C4-C5. Similar arenium intermediates can be hypothesized for the hydroxylation of 2-indanol.
Fig 4
Fig 4. E103G/F176A ToMO-catalyzed biotransformation of 2-indanol.
HPLC chromatogram at λ = 280 nm of an aliquot of the exhausted medium deriving from the 16 hrs, E103G/F176A ToMO-catalyzed biotransformation of 2-indanol.
Fig 5
Fig 5. Immunofluorescence assay.
Effect of sodium arsenite on the formation of stress granules: immunofluorescence and statistical analysis. PABP mAb and Cy3-conjugated goat anti-mouse F(ab’)2 were used to detect SGs in H9c2 cells cultured under normal growth (A) and SA-stress conditions (B). Nuclei are stained with DAPI. Scale bars: 10μm. (C) Effect of pretreatment with hydroxylated compounds in SA-treated H92c cardiomyoblasts. The table reports SGs number for cell (Mean ± SEM) that was obtained by using “Analyze Particles” plugin of the image software ImageJ (pixel unit: 1,5–2,0; Circularity: 0,99–1,00). Statistical analysis (unpaired t test, two tailed; (*, p ≤ 0.05; **, p ≤ 0.01). Tyr: tyrosol; Hyd: hydroxytyrosol.
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References

    1. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod. Biol Endocrinol. 2005;3:28–48. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1215514/. - PMC - PubMed
    1. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–399. Available: http://www.annualreviews.org/doi/abs/10.1146/annurev.arplant.55.031903.1.... - DOI - PubMed
    1. Marco S, Rullo R, Albino A, Masullo M, De Vendittis E, Amato M. The thioredoxin system in the dental caries pathogen Streptococcus mutans and the food-industry bacterium Streptococcus thermophilus. Biochimie. 2013;95(11):2145–56. Available: http://www.ncbi.nlm.nih.gov/pubmed/23954859. 10.1016/j.biochi.2013.08.008 - DOI - PubMed
    1. Anderson P, Kedersha N. Stressful initiations. J Cell Sci. 2002;115:3227–34. Available: http://jcs.biologists.org/content/115/16/3227.long. - PubMed
    1. Conti V, Corbi G, Russomanno G, Simeon V, Ferrara N, Filippelli W, et al. Oxidative stress effects on endothelial cells treated with different athletes' sera. Med Sci Sports Exerc 2012; 44(1):39–49. Available: http://journals.lww.com/acsm-msse/pages/articleviewer.aspx?year=2012&iss.... 10.1249/MSS.0b013e318227f69c - DOI - PubMed

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Grants and funding

This research was supported by FARB 2013 ORSA 132082. Dr. C. Sarcinelli was supported by grant POR CAMPANIA FSE 2007/2013, Progetto CARINA CUP B25B0900080007. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.