Optimized oxidoreductases for medium and large scale industrial biotransformations
CLOSE
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
CLOSE
Private area
User:


Password:

publications
Total records: 126
Pages:    1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  

[ 2016 ] van Kuijk SJA, del Río JC, Rencoret J, Gutiérrez A, Sonnenberg ASM, Baars JJP, Hendriks WH, Cone JW Selective ligninolysis of wheat straw and wood chips by the white-rot fungus Lentinula edodes and its influence on in vitro rumen degradability J. Anim. Sci. Biotechnol., 7: 55
[ 2016 ] Viña-Gonzalez J, González-Pérez D, Alcalde M Directed evolution method in Saccharomyces cerevisiae: Mutant library creation and screening J. Vis. Exp., doi: 10.3791/53761
[ 2015 ] Alcalde M Engineering the ligninolytic enzyme consortium Trends Biotechnol., 33: 155-162
[ 2015 ] Babot ED, del Río JC, Cañellas M, Sancho F, Lucas F, Guallar V, Kalum L, Lund H, Gröbe G, Scheibner K, Ullrich R, Hofrichter M, Martínez AT, Gutiérrez A Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study Appl. Environ. Microbiol., doi: 10.1128/AEM.00660-15
[ 2015 ] Babot ED, del Río JC, Kalum L, Martínez AT, Gutiérrez A Regioselective Hydroxylation in the Production of 25-Hydroxyvitamin D by Coprinopsis cinerea Peroxygenase ChemCatChem, 7: 283-290
[ 2015 ] Baratto MC, Sinicropi A, Linde D, Saez-Jimenez V, Sorace L, Ruiz-Dueñas FJ, Martínez AT, Basosi R, Pogni R Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study J. Phys. Chem. B, 119: 13583-13592
year2015
Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase
Ferreira P, Hernández-Ortega A, Lucas F, Carro J, Herguedas B, Borrelli K, Guallar V, Martínez AT, Medina M
FEBS J., 282: 3091-3106

Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2 O2 for lignin degradation at the expense of benzylic and other π system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping-pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping-pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state Kd and steady-state Km increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in AAO.

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