Lignin peroxidase L3 from Phlebia radiata. Pre-steady-state and steady-state studies with veratryl alcohol and a non-phenolic lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol.
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 beta-O-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 C alpha-C beta 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 H2O2, 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 II, respectively. Similar results have been reported for the LiP from Phanerochaete chrysosporium upon reaction with H2O2. However, compound I of L3 was more stable in the absence of additional electron donors. The second-order rate constant of compound I formation by H2O2 was determined to be 6 x 10(5) M-1 s-1 and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound II and further to native enzyme when either veratryl alcohol or the beta-O-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 II formation was similar with either veratryl alcohol or the beta-O-4 dimer (6.7 x 10(3) and 6.5 x 10(3) M-1 s-1, respectively). At pH 3.0 formation of compound II 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 beta-O-4 dimer involving both compounds I and II as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound II. Chemical evidence of such a radical, formed after the initial LiP-catalysed one-electron oxidation of beta-O-4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A. & Brunow, G. (1993) Holzforschung, 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.
Lundell
T
T
Department of Applied Chemistry and Microbiology, University of Helsinki, Finland.
Wever
R
R
Floris
R
R
Harvey
P
P
Hatakka
A
A
Brunow
G
G
Schoemaker
H
H
D001592:Benzyl Alcohols / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005658:Fungi / Q000201:enzymology*; D006140:Guaifenesin / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D010544:Peroxidases / Q000378:metabolism*; D013053:Spectrophotometry
D001592:Benzyl Alcohols; D007527:Isoenzymes; D046911:Macromolecular Substances; C047159:veratrylglycerol-beta-guaiacyl ether; D006140:Guaifenesin; D010544:Peroxidases; C042858:lignin peroxidase; C042197:veratryl alcohol