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Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin

Veronica Saez Jimenez (Institutionen för biologi och bioteknik, Industriell bioteknik) ; J. Rencoret ; M. A. Rodriguez-Carvajal ; A. Gutierrez ; F. J. Ruiz-Duenas ; A. T. Martinez ; Applied Marero S ; V. P. Environmental Microbiology
Biotechnology for Biofuels (1754-6834). Vol. 9 (2016),
[Artikel, refereegranskad vetenskaplig]

Background: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility. Results: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two-and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k(3app) values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have similar to 20-30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used. Conclusions: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.

Nyckelord: Ligninolytic peroxidases, Single-electron transfer, Catalytic tryptophan, Directed mutagenesis, site-directed mutagenesis, eryngii versatile peroxidase, substrate, interaction sites, phanerochaete-chrysosporium, pleurotus-eryngii, microbial-degradation, degrading peroxidases, escherichia-coli, veratryl, alcohol, cation radicals

Denna post skapades 2016-10-14. Senast ändrad 2016-10-14.
CPL Pubid: 243393


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Institutionen för biologi och bioteknik, Industriell bioteknik


Industriell bioteknik

Chalmers infrastruktur