How ligninolytic enzymes attack lignin: A rapid spectrophotometry and 2D NMR study using lignin permethylation and protein directed mutagenesis

Oespite they are claimed 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. Two water-soluble sulfonated lignins (from Picea abies and fucalyptus grandis) were chemically characterized and used to estimate single electrontransfer rates to the HzOz-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and 11, bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound 11 was quantified by stopped-flow rapid spectrophotometry, from 4-fold (softwood lignin) to over lOO-fold (hardwood lignin) lower electron-transfer efficiencies (k3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~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 t ransfer to the W164S compound 11 was found. This revea/ed that the residual reduction of W164S compound 11 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 20 NMR, 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. Interestingly, similar results were obtained for Phanerochaete chrysosporium LiP, the most thoroughly investigated ligninolytic enzyme. In this study, we demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin po/ymer.