Background Although lignin peroxidase is claimed as a key enzyme in enzyme-catalyzed lignin degradation in vitro enzymatic degradation of lignin was not easily observed in lab-scale experiments. made up of W251 residue was newly suggested based on the observation of repressed radical coupling and amazingly lower electron transfer rate for W215A mutant. Furthermore the retardation of the suicidal radical coupling between the W251 residue Gata1 and the monolignolic radical was attempted by supplementing the acidic microenvironment round the W251 residue to engineer radical-robust LiPH8. Among many mutants mutant A242D showed exceptional catalytic performances by yielding 21.1- BKM120 and 4.9-fold higher increases of kcat and kcat/KM values respectively in the oxidation of non-phenolic model lignin dimer. Conclusions A mechanism-based suicide inhibition of LiPH8 by phenolic compounds was firstly revealed and investigated in this work. Radical-robust LiPH8 was also successfully designed by manipulating BKM120 the transient radical state of radical-susceptible electron-relay. Radical-robust LiPH8 will play an essential role in degradation of lignin which will be BKM120 consequently linked with improved production of sugars from lignocellulose biomass. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0664-1) contains supplementary material which is available to authorized users. harbors uncovered catalytic W171 site which was demonstrated to play a vital role in the oxidation of high-redox potential substrates such as veratryl alcohol (VA) or non-phenolic lignin derivatives. The oxidation was manipulated through a long-range electron transfer (LRET) to the heme (for both compound I and compound II intermediates) [3]. The unique roles of the surface-active site in the oxidation of high-redox potential substrates or heavy lignin macromolecules BKM120 were also investigated for VP from were reported to BKM120 be improved through studies of an ancestral mutation method or comparative structural analysis [5 6 Besides those limitations the inhibitor conversation between the enzyme and the phenolic compound was emphasized as a significant factor which disrupts LRET and catalytic turnover of non-phenolic lignin dimer [7]. In this study the enzyme mechanism-based inhibition mode of the phenolic compound was investigated. The site responsible for the irreversible conversation between LiPH8 and free hydroxyl monolignol was searched by LC-MS/MS analysis. Surprisingly the W251 site was identified as a suicide site by coupling with the guaiacol radical (the product released from your degradation of VE dimer) and proved to be an essential electron-relay residue around the LRET route from your surface-active site W171 to heme. Its role as a stepping stone in the hopping ET mechanism was exhibited through the rational mutagenesis of its aromatic character. Creating an acidic environment round the radical coupling site to prevent coupling with the phenoxy radical was also examined for the rational design of effective LiP. With this purpose a combination of liquid chromatography-tandem mass spectrometry stopped-flow spectrophotometry and rational mutagenesis techniques was used. As BKM120 far as we know this is the first successful trial to increase the catalytic overall performance of LiPH8 by altering the intramolecular ET route from the surface site to heme. Methods Materials Hydrogen peroxide hemin oxidized glutathione ampicillin isopropyl-b-d-thiogalactopyranoside 2 2 (3-ethylbenzothiazoline-6-sulfonate) (ABTS) guanidine hydrochloride dibasic potassium phosphate citric acid trizma hydrochloride and guaiacol used in this study were purchased from your Sigma Chemical Co. South Korea and were used without any further purification. Veratrylglycerol-beta-guaiacyl ether (VE dimer) at 97% purity was obtained from AstaTech Inc. USA. Recombinant enzyme preparation The LiPH8 synthetic gene including the seven-residue pro-sequence was synthesized by the Bioneer Organization (South Korea). The gene coding protein sequence was retrieved from a previously published statement [8] (UniProtKB access: “type”:”entrez-protein” attrs :”text”:”P06181″ term_id :”126285″ term_text :”P06181″P06181). The refolding and purification procedures were performed as previously reported [8]. The mutant LiPH8 genes were constructed using a.