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

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 kcat/Km for the salicylate reaction catalyzed by the holo form is \u3e105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat 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

Crystallographic structure and molecular dynamics simulations of the major endoglucanase from Xanthomonas campestris pv. campestris shed light on its oligosaccharide products release pattern

Cellulases are essential enzymatic components for the transformation of plant biomass into fuels, renewable materials and green chemicals. Here, we determined the crystal structure, pattern of hydrolysis products release, and conducted molecular dynamics simulations of the major endoglucanase from the Xanthomonas campestris pv. campestris (XccCel5A). XccCel5A has a TIM barrel fold with the catalytic site centrally placed in a binding groove surrounded by aromatic side chains. Molecular dynamics simulations show that productive position of the substrate is secured by a network of hydrogen bonds in the four main subsites, which differ in details from homologous structures. Capillary zone electrophoresis and computational studies reveal XccCel5A can act both as endoglucanase and licheninase, but there are preferable arrangements of substrate regarding β-1,3 and β-1,4 bonds within the binding cleft which are related to the enzymatic efficiency136493502CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP405191/2015-4; 303988/2016-9; 440977/2016-9; 151963/2018-5; 490022/2009-010/52362-5; 11/20505-4; 11/21608-1; 15/50590-4; 15/13684-0; 2009/52840-7This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) via grants 10/52362-5, 11/20505-4, 11/21608-1, 15/50590-4 and 15/13684-0; INCT Bioetanol (FAPESP/CNPq); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) via grants 405191/2015-4, 303988/2016-9, 440977/2016-9 and 151963/2018-5 and the MCT/CNPq/FAPESP EU-Brazil Collaboration program in Second Generation Biofuels (CeProBio Project; FAPESP 2009/52840-7 and CNPq 490022/2009-0