2024-03-28T15:38:36Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1235162021-12-27T16:24:57Zcom_10261_86com_10261_1com_10261_15com_10261_6col_10261_339col_10261_268
Demonstration of lignin-to-peroxidase direct electron transfer: A transient-state kinetics, directed mutagenesis, EPR, and NMR study
Sáez-Jiménez, Verónica
Baratto, M. Camila
Pogni, Rebecca
Rencoret, Jorge
Gutiérrez Suárez, Ana
Santos, José Ignacio
Martínez, Ángel T.
Ruiz-Dueñas, F. J.
Ministerio de Economía y Competitividad (España)
European Commission
Amino acids
Electron spin resonance spectroscopy
Electron transitions
Excited states
Free radical reactions
Hardwoods
Lignin
Manganese
Molecular mass
Mutagenesis
Nuclear magnetic resonance spectroscopy
Oxidation
Rate constants
Redox reactions
Size exclusion chromatography
Spectrophotometry
14 páginas.-- 10 figuras.-- 1 tabla.-- 46 referencias.-- Author's Choice—Final version free via Creative Commons CC-BY license.
Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn2 , and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan
2015-09-18
artículo
Journal of Biological Chemistry Journal of Biological Chemistry 290(38): 23201-23213 (2015)
0021-9258
http://hdl.handle.net/10261/123516
10.1074/jbc.M115.665919
1083-351X
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100000780
26240145
eng
Publisher's version
http://dx.doi.org/10.1074/jbc.M115.665919
Sí
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL2014-53730-R
http://creativecommons.org/licenses/by/3.0/
openAccess
American Society for Biochemistry and Molecular Biology