Lignin peroxidase L3 from Phlebia rediata

Abstract

The catalytic cycle of lignin peroxidase (Lip, ligninase) isozyme L3 from the white-rot fungus Phlebia radiata was investigated using stopped-flow techniques. Veratryl (3,4-dimethoxybenzyl) alcohol and a lignin model compound, non-phenolic a-0-4 dimer 1 -(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, were used as electron donors. This is the first report on the detailed kinetic analysis of a Lip-catalysed Ca-CB bond cleavage of the dimer, representing the major depolymerisation reaction in the lignin polymer. The native enzyme showed a typical heme peroxidase absorbance spectrum with a Soret maximum at 407 nm. Following the reaction with H,Oz, the Soret band decreased in absorbance, shifted to 403 nm and then to 421 nm, demonstrating the formation of compound I followed by the formation of compound 11, respectively. Similar results have been reported for the Lip from Phunerochaete chrysosporium upon reaction with H,O,. However, compound I of L3 was more stable in the absence of additional electron donors. Thesecond-order rate constant of compound I formation by H,O, was determined to be 6 X IOI M-' s-' and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound Il and further to native enzyme when either veratryl alcohol or the p-0-4 dimer was supplied as electron donor and in both cases veratraldehyde appeared as the major product. At pH 6.0, the second-order rate constant for compound I1 formation was similar with either veratryl alcohol or the p-0-4 dimer (6.7 X lo3 and 6.5 X 103 M-' s-', respectively). At pH 3.0 formation of compound I1 with either reductant proceeded so rapidly that determination of the respective rate constants was not possible. The results point to identical catalytic cycles of L3 with veratryl alcohol or the p-0-4 dimer involving both compounds I and I1 as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound 11. Chemical evidence of such a radical, formed after the initial Lip-catalysed one-electron oxidation of p-0-4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A. & Brunow, G. (1993) Holgorschung, in the press]. The catalytic redox-cycle and the oxidation mechanism presented here reconcile seemingly contradictory results obtained in previous studies on Lip kinetics during the last decade.Lignin peroxidase (Lip, ligninase) is an extracellular lignin-degrading enzyme, first discovered in the white-rot fungus Phanerochaete chrysosporium Burds. [I, 21. The enzyme is a glycoprotein that typically has molecular mass of

Reaction Kinetics of Versatile Peroxidase for the Degradation of Lignin Compounds

“…In contrast, Lundell et al (1993b) and Schoemaker et al (1994b) concluded a slightly different non-enzymatic reaction after Cα-Cβ cleavage assuming structure II (in Fig. 2) with a cation radical centre at the Cβ-ether oxygen position as consequence of LiP action.…”

“…Here, the catalytic cycle will be initiated again by H 2 O 2 to form E I . E I further reacts with adlerol (structure I) to E II and an adlerol cation radical intermediate (structure II) (Lundell et al, 1993b) which will be rapidly fragmented non-enzymatically between its Cα-Cβ bonds in the subsequent (Hatakka et al, 1991). For the latter, various theories have been under consideration.…”

Participation of Mn(II) in the catalysis of laccase, manganese peroxidase and lignin peroxidase from Phlebia radiata

“…Furthermore, the main J? radiata LIP isozyme L3 is kinetically similar to p chrysosporium LIP H8 [9]. The characterized R radiata MnP also resembles biochemically the E!…”

“…Extracellular proteins were separated by FPLC anion-exchange chromatography at pH 5.5 on Sepharose Q (Pharmacia Biotechnology) as described previously [2,9]. Enzyme activities of protein fractions were analyzed and the peak fractions showing only laccase, MnP or LiP activity were pooled and concentrated (10 kDa cut-off Microsep concentrators, Filtron).…”

Lignin Degradation Processes and the Purification of Valuable Products

“…These enzymes are relatively nonspecific, and can therefore oxidize phenolic aromatic substrates as well as various non-phenolic lignin model compounds [59][60][61]. The importance of lignin peroxidases for lignin depolymerization reflects their ability to catalyze the cleavage of α,β and β-ether bonds (including β-O-4 linkages) leading to the efficient degradation of lignin into mono-aromatic structures, which has been demonstrated using lignin model compounds [61].…”