Catalytic Mechanism for the Conversion of Salicylate Into Catechol by the Flavin-Dependent Monooxygenase Salicylate Hydroxylase

Authors

Débora M. A. Costa, Universidade Federal de Minas Gerais
Stefanya V. Gómez, Universidade Federal de Minas Gerais
Simara S. de Araújo, Universidade Federal de Minas Gerais
Mozart S. Pereira, Universidade Federal de Minas Gerais
Rosemeire B. Alves, Universidade Federal de Minas Gerais
Denize C. Favaro, Universidade Estadual de Campinas
Alvan C. Hengge, Utah State UniversityFollow
Ronaldo A. P. Nagem, Universidade Federal de Minas Gerais
Tiago A. S. Brandão, Universidade Federal de Minas Gerais

Document Type

Article

Journal/Book Title

International Journal of Biological Macromolecules

Publication Date

1-28-2019

Publisher

Elsevier

Volume

129

First Page

1

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Last Page

67

Abstract

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The k_cat/K_m for the salicylate reaction catalyzed by the holo form is >10⁵ M⁻¹ s⁻¹ at pH 8.5 and 25 °C. Hammett plots for K_m and k_cat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

Recommended Citation

Costa, Débora M.A., et al. “Catalytic Mechanism for the Conversion of Salicylate into Catechol by the Flavin-Dependent Monooxygenase Salicylate Hydroxylase.” International Journal of Biological Macromolecules, vol. 129, 28 Jan. 2019, pp. 588–600. doi: 10.1016/j.ijbiomac.2019.01.135