Reductive Dehalogenation of Aliphatic Halocarbons by Lignin Peroxidase of Phanerochaete chrysosporium

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.

The Role of Oxalate in Lignin Peroxidase-Catalyzed Reduction: Protection from Compound III Accumulation

Long-term objectives are to elucidate the role and mechanism of the various isozymes in lignin biodegradation. Work is described on electrochemical studies on lignin and Mn peroxidases. This study was performed to investigate the structural aspects which confer the lignin and Mn peroxidases with their high reactivity. The experimentally determined redox potential of the Fe{sup 3+}/Fe{sup 2+} couple for the lignin peroxidase isozymes H1, H2, H8 and H10 are very similar, near-130 mV. The redox potential for the Mn peroxidase isozymes H3 and H4 are similar to each other ({minus}88 mV and {minus}95 mV, respectively) and are more positive than the lignin peroxidases. The higher redox potential for the Fe{sup 3+}/Fe{sup 2+} couple is consistent with the heme active site of these fungal peroxidases being more electron deficient. To investigate the accessibility of the heme active site to the substrate which is oxidized [veratryl alcohol and Mn (II)], we investigated whether these substrates had any affect on the redox potential of the heme. The E{sub m7} value for lignin and Mn peroxidases are not affected by their respective substrates, veratryl alcohol and Mn (II). These results suggest that substrates do not directly interact with the ferric heme-iron as axial ligands. This is consistent with the present model for peroxidase catalysis. Suicide inhibitor (1) and nmr studies (2) indicate that the heme-iron of horseradish peroxidase (HRP) is not fully accessible to bulky substrates occur at the periphery of the heme.

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.

Lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA), a secondary metabolite of the fungus, to veratryl aldehyde (VAD). The oxidation of VA does not seem to be simply one-electron oxidation by LiP compound I (LiPI) to its cation radical (VA.+) and the second by LiP compound II (LiPII) to VAD. Moreover, the rate constant for LiPI reduction by VA (3 x 10(5) M-1 s-1) is certainly sufficient, but the rate constant for LiPII reduction by VA (5.0 +/- 0.2 s-1) is insufficient to account for the turnover rate of LiP (8 +/- 0.4 s-1) at pH 4.5. Oxalate was found to decrease the turnover rate of LiP to 5 s-1, but it had no effect on the rate constants for LiP with H2O2 or LiPI and LiPII, the latter formed by reduction of LiPI with ferrocyanide, with VA. However, when LiPII was formed by reduction of LiPI with VA, an oxalate-sensitive burst phase was observed during its reduction with VA. This was explained by the presence of LiPII, formed by reduction of LiPI with VA, in two different states, one that reacted faster with VA than the other. Activity during the burst was sensitive to preincubation of LiPI with VA, decaying with a half-life of 0.54 s, and was possibly due to an unstable intermediate complex of VA.+ and LiPII. This was supported by an anomalous, oxalate-sensitive, LiPII visible absorption spectrum observed during steady state oxidation of VA. The first order rate constant for the burst phase was 8.3 +/- 0.2 s-1, fast enough to account for the steady state turnover rate of LiP at pH 4.5. Thus, it was concluded that oxalate decreased the turnover of LiP by reacting with VA.+ bound to LiPII. The VA.+ concentration measured by electron spin resonance spectroscopy (ESR) was 2.2 microM at steady state (10 microM LiP, 250 microM H2O2, and 2 mM VA) and increased to 8.9 microM when measured after the reaction was acid quenched. Therefore, we assumed the presence of two states of VA.+ bound to LiPII, one ESR-active and one ESR-silent. The ESR-silent species, which could be detected after acid quenching, would be responsible for the burst phase. Both of the VA.+ species disappeared in the presence of 5 mM oxalate. The ESR-active species reached a maximum (3.5 microM) at 0.5 mM VA under steady state. From these studies, a mechanism for VA oxidation by LiP is proposed in which a complex of LiPII and VA.+ reacts with an additional molecule of VA, leading to veratryl aldehyde formation.

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. In this work, we investigated the roles of veratryl alcohol and lignin in the ligninolytic system of P. chrysosporium BKM-F-1767 cultures grown under nitrogen-limited conditions. Cultures supplemented with 0.4 to 2 mM veratryl alcohol showed increased lignin peroxidase activity. Addition of veratryl alcohol had no effect on Mn-dependent peroxidase activity and inhibited glyoxal oxidase activity. Azure-casein analysis of acidic proteases in the extracellular fluid showed that protease activity decreased during the early stages of secondary metabolism while lignin peroxidase activity was at its peak, suggesting that proteolysis was not involved in the regulation of lignin peroxidase activity during early secondary metabolism. In cultures supplemented with lignin or veratryl alcohol, no induction of mRNA coding for lignin peroxidase H2 or H8 was observed. Veratryl alcohol protected lignin peroxidase isozymes H2 and H8 from inactivation by H2O2. We conclude that veratryl alcohol acts as a stabilizer of lignin peroxidase activity and not as an inducer of lignin peroxidase synthesis.

• Genes for ligninolytic enzymes, normally associated with white-rot fungi, are shown to be widespread in a broad taxonomic range of ectomycorrhizal (ECM) fungi. • ECM fungi were screened for lignin peroxidase (LiP) and manganese peroxidase (MnP) genes by PCR using primers specific for known isozymes in the white-rot fungus Phanerochaete chrysosporium, with DNA sequencing used to confirm the identity of the amplified fragments. • Genes for LiPs were detected in ECM fungi representing the orders Agaricales, Aphyllophorales, Boletales, Cantharellales, Hymenochaetales, Sclerodermatales, Stereales and Thelephorales. MnP genes were detected in only Cortinarius rotundisporus and three ECM Stereales taxa. • The presence of genes for decomposer activities supports putative evolutionary relationships between ECM and saprotrophic fungi. Expression of the lignolytic genes may facilitate ECM fungal access to nutrients associated with dead plant material in soil and potentially a supplementary carbon supply. Strict functional boundaries between ECM and decomposer fungi may be less clear-cut than previously thought.

Degradation of tetracycline and oxytetracycline by crude lignin peroxidase prepared from Phanerochaete chrysosporium – A white rot fungus

Lignin peroxidase H2 from Phanerochaete chrysosporium: Purification, characterization and stability to temperature and pH

This is a continuation of our previous paper on production of lignin peroxidase (LiP) by Phanerochaete chrysosporium in solid substrate fermentation (SSF) medium of corncobs. The enzyme was purified by ammonium sulphate precipitation and ion-exchange fast protein liquid chromatography. Maximum yield of LiP was 13.7 U/gds (units per gram dry substrate) after 5 days of SSF with 70% moisture and 20% (v/w) inoculum. The approximate molecular mass of purified LiP, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 38 kDa. The pH and temperature optima for the LiP were 4 and 40°C, respectively. Immobilization of LiP in hydrophobic xerogels caused hyperactivation of LiP and enhanced its thermostability properties. The KM and Vmax values for immobilized LiP were 10.56 mg/ml and 16.67 μmol/min (120.49 U/mg of protein) as compared to 13 mg/ml and 11.76 μmol/min (85 U/mg of protein), respectively, for free LiP using veratryl alcohol as substrate.

We demonstrate direct oxidation of ferrocytochrome c by lignin peroxidase (LiP) from the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Steady-state kinetic data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism, suggesting that the reductions of LiP compounds I and II by ferrocytochrome c are irreversible. The pH dependence of the overall reaction apparently is controlled by two factors, the pH dependence of the electron-transfer rate and the pH dependence of enzyme inactivation in the presence of H2O2. In the presence of 100 microM H2O2, veratryl alcohol (VA) significantly enhanced cytochrome c oxidation at pH 3.0 but had little effect above pH 4.5. In the presence of < 10 microM H2O2, the stimulating effect of VA on the reaction is greatly diminished. As with cytochrome c peroxidase reactions, LiP oxidation of ferrocytochrome c decreased as the ionic strength increased, implying the involvement of electrostatic interactions between the polymeric substrate and enzyme. The reaction product ferricytochrome c inhibited VA oxidation by LiP in a noncompetitive manner, suggesting that cytochrome c binds to LiP at a site different from the small aromatic substrate binding site. Recent crystallographic studies show that the heme is buried in the LiP protein and unavailable for direct interaction with polymeric substrates, suggesting that electron transfer from ferrocytochrome c to LiP occurs over a relatively long range. The role of VA in this electron-transfer reaction is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)