Optimized oxidoreductases for medium and large scale industrial biotransformations
Project Secretariat
Dr Marta Pérez-Boada
E-mail: MPBoada@cib.csic.es
Consejo Superior de Investigaciones Científicas (CSIC)
Biological Research Centre (CIB)
Calle Ramiro de Maeztu 9, E-28040 Madrid, Spain
Phone: 34 918373112
Fax: 34 915360432
Mobile: 34 650080476
Private area


Total records: 116
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[ 2014 ] Carro J, Ferreira P, Rodríguez L, Prieto A, Serrano A, Balcells B, Ardá A, Jiménez-Barbero J, Gutiérrez A, Ullrich R, Hofrichter M, Martínez AT 5-Hydroxymethylfurfural conversion by fungal aryl-alcohol oxidase and unspecific peroxygenase FEBS J., 282: 3218-3229
[ 2014 ] Fernandez-Fueyo E, Acebes S, Ruiz-Dueñas FJ, Martínez MJ, Romero A, Medrano FJ, Guallar V, Martínez AT Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi Acta Crystal. D, 70: 3253-3265
[ 2014 ] Fernandez-Fueyo E, Castanera ER, Ruiz-Dueñas FJ, López-Lucendo MF, Ramírez L, Pisabarro AG, Martínez AT Ligninolytic peroxidase gene expression by Pleurotus ostreatus: Differential regulation in lignocellulose medium and effect of temperature and pH Fungal Gen. Biol., doi: 10.1016/j.fgb.2014.02.003
[ 2014 ] Fernandez-Fueyo E, Ruiz-Dueñas FJ, Martínez AT Engineering a fungal peroxidase that degrades lignin at very acidic pH Biotechnol. Biofuels, 7: 114
[ 2014 ] Fernandez-Fueyo E, Ruiz-Dueñas FJ, Martínez MJ, Romero A, Hammel KE, Medrano FJ, Martínez AT Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability Biotechnol. Biofuels, 7: 2
[ 2014 ] García-Ruiz E, Maté D, González-Pérez D, Molina-Espeja P, Camarero S, Martínez AT, Ballesteros A, Alcalde M Directed evolution of ligninolytic oxidoreductases: from functional expression to stabilization and beyond In "Cascade Biocatalysis. Integrating Stereoselective and Environmentally Friendly Reactions", First Edition. Edited by Sergio Riva and Wolf-Dieter Fessner. Wiley-VCH Verlag GmbH & Co
Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase
Carro J, Ferreira P, Martínez AT, Gadda G
Biochemistry, doi: 10.1021/acs.biochem.8b00106

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [α-2H2]-p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s−1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N−H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H+ transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H+ from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data resented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes. 

Official webpage of indox [ industrialoxidoreductases ]. Optimized oxidoreductases for medium and large scale industrial biotransformations. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement nº: FP7-KBBE-2013-7-613549. © indox 2013. Developed by garcíarincón