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At least 991 records · Page 55Linked to original sources
PubMed1990

Characterization of a new lignin peroxidase gene (GLG6) from Phanerochaete chrysosporium.

Sequence analysis of a new lignin peroxidase (LIP) gene, GLG6, from P. chrysosporium showed that it encodes a mature LIP protein with a predicted Mr of 36,454. A 28 aa signal peptide precedes the mature protein. The coding region of GLG6 is interrupted by nine introns ranging in size from 50-57 bp. GLG6 encoded-LIP has 72%, 88% and 82% homology, respectively, to the LIP isozymes H2, H3, and H10. Comparison of the N-terminal sequence of GLG6-encoded LIP to that of the LIP proteins H2, H8, H10 also showed that the former is relatively less related to the H2 protein than it is to the H8 and H10 proteins. Expression of GLG6, similar to the other LIP genes, occurs only during secondary metabolism.

Amino Acid Sequence↗
PubMed2001

On a revised mechanism of side product formation in the lignin peroxidase catalyzed oxidation of veratryl alcohol.

The O2-dependent formation of side products during the oxidation of veratryl alcohol (VA) by lignin peroxidase has previously been proposed to start with the attack of H2O on the VA radical cation (VA*+). This initial reaction is unlikely since it would also lead to side product formation in the absence of O2, which is not the case. In the current mechanism VA* reacts first with O2, whereafter H2O attacks. Furthermore, this paper describes an alternative explanation for the inhibitory effect of Mn2+ on VA side product formation. It is proposed that Mn2+ reduces reactive intermediates back to VA.

Benzyl Alcohols↗
PubMed1998

Purification and characterization of two lignin peroxidase isozymes produced by Bjerkandera sp. strain BOS55.

The white-rot fungus Bjerkandera sp. strain BOS55 excretes at least seven lignin peroxidase (LiP) isozymes. Two of these, LiP-2 and LiP-5 (molecular weight 40-42 kDa), were purified to homogeneity. Both isozymes had the same N-terminal amino acid sequence which showed strong homology with LiP isozymes produced by other white-rot fungi. The kinetics of both isozymes were similar. LiP-5 oxidized veratryl alcohol optimally only in the presence of H2O2 near pH 3.0 (16.7 U/mg) and LiP-2 did this below pH 2.5 (33.8 U/mg). Also at normal physiological pHs for fungal growth (pH 5.0-6.5) both isozymes were still active. Further characterization of LiP-2 and LiP-5 revealed that the Km for H2O2 strongly decreased with increasing pH. As a result of this the catalytic efficiency (TN/Km) calculated on the basis of the Km for H2O2 in the oxidation of veratryl alcohol was constant over wide pH range.

Amino Acid Sequence↗
PubMed1998

Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase.

In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O-acetyl esters were tested and were shown to be converted to O-acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.

Benzaldehydes↗
PubMed1994

Crystal structure of the fungal peroxidase from Arthromyces ramosus at 1.9 A resolution. Structural comparisons with the lignin and cytochrome c peroxidases.

The crystal structure of the peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) from the hyphomycete Arthromyces ramosus (ARP) has been determined by the multiple isomorphous replacement method and refined by the simulated annealing method to a crystallographic R-factor of 17.4% for the 19,191 reflections with F > 2 sigma F between 7.0 and 1.9 A resolution. The model includes residues 9 to 344, the heme group, two N-acetylglucosamine residues, two calcium ions and 246 water molecules. The root-mean-square deviation of bond lengths from the ideal values is 0.02 A. The mean coordinate error is estimated as 0.2 A. The electron density of the glycine-rich region of the amino-terminal eight residues was invisible. ARP has ten major and two short alpha-helices and a few short beta-strands. The overall tertiary structure of ARP is similar to that of yeast cytochrome c peroxidase (CCP) and is particularly similar to that of the lignin peroxidase (LiP) from Phanerochaete chrysosporium. Relative to CCP, ARP and LiP each have an extension of approximately 40 residues at the carboxy terminus. All eight cysteine residues in ARP form disulfide bonds (C12:C24, C23:C293, C43:C129 and C257:C322). Two calcium sites are inaccessible to solvent. The four disulfide bonds and two calcium sites, which are lacking in CCP, are conserved in ARP and LiP. The bond from Asn304C to Ala305N in ARP is the site sensitive to proteases. An Asx turn present in the Asn303 to Ala305 segment appears to orient the side-chain of Asn304 to outward from the molecule, rendering it easily trappable by pockets of proteases. The proximal heme ligand is His184 in helix F (distance of N epsilon 2 ... Fe, 2.10 A), and one of several water molecules in the distal pocket of the heme bridges the iron atom and the N epsilon 2 of His56. The orientation of the imidazole ring of the distal histidine residue relative to the heme group in ARP differs significantly from that in LiP. The access channel to the distal side of the heme of ARP is markedly wider along the heme plane than that of LiP. Many of the amino acid residues that comprise the entrance of this channel differ for ARP and LiP. This may account for the differences in substrate specificity.

Acetylglucosamine↗
PubMed2000

Chemo-enzymatic synthesis and characterization of graft copolymers from lignin and acrylic compounds.

Chemo-enzymatic initiation of graft copolymerization of acrylic compounds onto different technical lignosulfonates (LS) was compared to a Fenton-like system (ferrous ion, t-BHP). The enzyme tested was a phenoloxidase laccase (EC 1.10.3.2) from the white rot basidomycete Trametes versicolor. Most applied lignins were successfully grafted, resulting in a polymer yield of more than 90%. The effect of initiator concentration and the lignin/monomer ratio on the yield and M(w) of enzymatically grafted polymers were studied. The homopolymer proportion in the enzymatically produced grafts of Ca-LS and acrylic acid was 5 to 6x lower than those initiated by the Fenton-like reagent; no such differences were observed for Na-LS.

Journal Article↗
PubMed2001

Inactivation of lignin peroxidase during oxidation of the highly reactive substrate ferulic acid.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA) was found to be a highly reactive substrate for lignin peroxidase (LIP), exhibiting a k(cat) of 41.7 s(-1). Despite the high reactivity, two modes of inactivation prevailed during the oxidation of FA. The first, H(2)O(2)-dependent inactivation, was evidenced by incomplete substrate oxidation and accumulation of LIP compound III (LIPIII), even at relatively low H(2)O(2) concentrations. This was attributed to the high turnover rate along with the inability of FA to revert LIPIII to the native state, as evidenced by pre-steady-state kinetics. H(2)O(2)-dependent inactivation could be avoided by inclusion of veratryl alcohol (VA), which efficiently reverts LIPIII to the native state. However, VA also mediated FA oxidation, and significantly decreased the reaction rate, which is unlike for previously reported VA-mediated reactions. The second mechanism of LIP inactivation was attributed to binding of phenoxy radicals or oxidation products to the enzyme and its extent directly correlated with the amount of FA consumed. This inactivation could be considerably suppressed by inclusion of gelatin. Therefore, during the oxidation of highly reactive phenolics, different kinds of protectors are required for efficient oxidation and maintaining LIP activity over time. This is of importance when considering emerging biotechnological applications for LIP.

Journal Article↗
PubMed2000

Metabolism of cellulose by Phanerochaete chrysosporium in continuously agitated culture is associated with enhanced production of lignin peroxidase.

Production of the extracellular heme protein lignin peroxidase (LiP) by Phanerochaete chrysosporium is currently associated with a number of requirements, namely exposure of the cultures to oxygen; limiting nutrient nitrogen or carbon and static or semi-static culture conditions. To obtain LiP activity in continuously agitated liquid culture requires the inclusion of a surfactant. However, using cellulose as the carbon source, we obtained high titres (0.2-0.4 U ml(-1)) of LiP in submerged liquid cultures under conditions of continuous agitation, without substrate limitation or the need to add oxygen or surfactant. Comparison of the morphological and physiological traits of hyphae maintained on either cellulose or free glucose supports observations that the synthesis of extracellular polysaccharide in the cultures grown on glucose, restricts oxygen diffusion into the hyphae, which is necessary for LiP induction. They also suggest that isozymes of LiP synthesised under these conditions may be triggered in response to oxidant stress.

Cellulose↗
PubMed2001

The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium.

Convective Interaction Media (CIM) monolithic columns were applied for the HPLC monitoring of Phanerochaete chrysosporium lignin peroxidase (LiP) isoforms during cultivation. The influence of the agitation mode (circular, elliptic) and rate (130 and 200 rpm), as well as the initial nitrogen concentration (1.6-6 mM) in the growth medium was investigated. Identical rotation rate but different agitation modes resulted in different LiP activities and isoenzyme compositions. On the other hand, at different agitation types and rates, similar LiP activities were obtained at different isoenzyme compositions. Although LiP H2 and LiP H6/H7 were predominant isoenzymes obtained at various cultivation conditions, relative isoenzyme amounts differ considerably when initial nitrogen concentration was changed between 1.6 and 5 mM.

Culture Media↗
PubMed1997

Removal of lignin and tannin colour from aqueous solution by adsorption onto activated charcoal.

Sorptive uptake of lignin and tannin from an aqueous phase by activated charcoal was investigated in the laboratory. The sorption reaction was found to be of a first order. The influence on the rate of sorption of various factors, such as amount of sorbent and pH of the system, have been investigated. Sorption data fit well into the Langmuir adsorption isotherm, indicating formation of a monolayer over a homogeneous sorbent surface. Sorption capacity, rate constant, intraparticle diffusion coefficient, etc. were calculated from the sorption data. Desorption studies indicate the irreversible nature of the sorption reaction, whereas interruption studies suggest film diffusion to be rate limiting.

Journal Article↗
PubMed1995

Evidence for formation of the veratryl alcohol cation radical by lignin peroxidase.

Lignin peroxidases (LiP) catalyze the H2O2-dependent two-electron oxidation of veratryl alcohol (VA) to veratryl aldehyde. We present here, electron spin resonance (ESR) evidence for the formation of the one-electron oxidized intermediate, the veratryl alcohol cation radical (VA.+). The ESR spectrum of VA.+ was first obtained in a fast-flow system with Ce(IV) as an oxidant and 10% HNO3 to stabilize the radical. This ESR signal was deconvoluted, and the hyperfine splitting constants were determined. The identity of the radical was confirmed by computer simulation of the ESR spectrum and calculation of spin and charge densities on the radical. An identical radical signal was observed with LiP, also in a fast-flow incubation containing 10 microM LiP, 2 mM VA, and 500 microM H2O2 at pH 3.5. The Fourier transforms of the ESR signals further confirmed that the spectra obtained with both Ce(IV) and LiP were due to the same radical species. The VA.+ had a distinct visible spectrum in 98% H2SO4 with an absorbance maximum at 529 nm. The extinction coefficient of the VA.+ spectral band at 529 nm was calculated to be 11,000 M-1 cm-1. The VA.+ was found to be a strong acid, as are other cation radicals, with the pKa at -1.0 pH. This value was determined by quantitating both the concentration of VA.+ by visual and ESR spectrometry and the g-value of the ESR signal at various pH values.

Benzyl Alcohols↗
PubMed1995

The effect of manganese on the oxidation of chemicals by lignin peroxidase.

It has recently been discovered that lignin peroxidase isozyme H2 (LiPH2) has the ability to oxidize Mn2+ (Khindaria et al., 1995). Furthermore, at pH 4.5, the physiological pH of Phanerochaete chrysosporium, LiPH2 oxidizes Mn2+ at a much faster rate (25 times) than veratryl alcohol (VA). The ability of Mn2+ to act as a redox mediator for indirect oxidations catalyzed by LiPH2 was therefore investigated. In the presence of physiologically relevant levels of oxalate and Mn2+, the rate of LiPH2-catalyzed oxidation of all substrates studied was dramatically increased. Up to 10-fold stimulations were observed compared to the rates of oxidation of substrate in either the presence or absence of VA. We propose that the stimulation is due to the ability of LiPH2 to oxidize Mn2+, producing the strong oxidant Mn3+, at a high rate. The rates of oxidation of the substrates showed a hyperbolic dependence on Mn2+ in the presence of oxalate, a chelator which was required for maximal activity. The oxalate dependence of the oxidation rates correlated well with the concentration of the 1:1 complex of Mn(2+)-oxalate. The relative concentrations of the substrates and H2O2 and the rate constants for their reactions with Mn3+ determined which chemical was oxidized by the enzymatically produced Mn3+. The importance of the ability of Mn(2+)-oxalate to stimulate the oxidation of chemicals by LiPH2 is discussed.

Basidiomycota↗
PubMed1995

X-ray absorption spectroscopy comparison of the active site structures of Phanerochaete chrysosporium lignin peroxidase isoenzymes H2, H3, H4, H5, H8, and H10.

The iron heme and its immediate environment can provide information that is pivotal to our understanding of the structural and mechanistic features that confer unusual properties to the heme peroxidases. X-ray absorption spectroscopy (XAS), which is ideally suited for the investigation of the local environment and electronic structure of the heme iron of hemeproteins, has been used to characterize a variety of lignin peroxidase and manganese-dependent peroxidase isoenzymes produced by the white rot fungus Phanerochaete chrysosporium. The data suggest no differences within the error in the first coordination shell of iron for the isoenzymes H2, H3, H4, H5, H8, and H10 examined in this study. The pyrrole nitrogens are at a distance of 2.05 +/- 0.015 A, and the proximal histidine nitrogens are at 1.93 +/- 0.02 A, while the sixth ligands are located at 2.17 +/- 0.03 A. Significant differences are observed in higher coordination shells which may be related to conformational differences in the heme.

Binding Sites↗
PubMed1994

Oxidation of dibenzo-p-dioxin by lignin peroxidase from the basidiomycete Phanerochaete chrysosporium.

Dibenzo-p-dioxin (I) was rapidly degraded in ligninolytic cultures of the basidiomycete Phanerochaete chrysosporium. Lignin peroxidase (LiP) oxidized I to generate the following products: catechol (V), dibenzo-p-dioxin-2,3-quinone (VIII), 2-hydroxy-5-(2-hydroxyphenoxy)-1,4-benzoquinone (IX), 4,5-dihydroxy-1,2-benzoquinone (X), 2-(2-hydroxyphenoxy)-1,4-benzoquinone (XI), 4-hydroxy-1,2-benzoquinone (XII), and 1,2-benzoquinone (XIII). Identical products were formed when the reaction was conducted under argon. No incorporation of 18O into products was observed when the reaction was conducted under 18O2. Oxidation of I in H(2)18O resulted in incorporation of two atoms of 18O into the quinone VIII. Nonenzymatic hydrolysis of the quinone (VIII) yielded catechol (V), IX and X. Hydrolysis of VIII in H(2)18O resulted in incorporation of 18O atoms into IX and X, whereas no incorporation of 18O atoms into V was observed. These results are explained by mechanisms involving the one-electron oxidation of I by LiP to produce the corresponding cation radical. Nucleophilic attack of water on the cation radical generates a 2-hydroxydibenzo-p-dioxin radical, which is oxidized to a delocalized cation. The attack of water at position C-4a of the 2-hydroxydibenzo-p-dioxin cation, followed by oxidation and C-O-C bond cleavage, lead to formation of the quinone (XI), which undergoes 1,4-addition of water and cleavage of the second C-O-C bond to generate V and XII.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗
PubMed1993

NMR investigation of isotopically labeled cyanide derivatives of lignin peroxidase and manganese peroxidase.

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.

Basidiomycota↗
PubMed2002

Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L.

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.

Benzyl Alcohols↗
PubMed1996

EPR detection and characterization of lignin peroxidase porphyrin pi-cation radical.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes the H2O2 dependent one- and two-electron oxidations of substrates. The catalytic cycle involves the oxidation of ferric-LiP by H2O2 by two electrons to compound I, which is an oxoferryl heme and a free radical. It has been speculated that the unpaired electron is in a pi delocalized porphyrin radical. However, no direct evidence for the presence of the free radical has been reported. We present electron paramagnetic resonance (EPR) detection and characterization of compound I of LiP. The LiP compound I EPR signal is different than those reported previously for compound I of horseradish peroxidase and chloroperoxidase. However, the EPR signal of compound I of LiP (axial g tensor extending from gperpendicular = 3.42 to gparallel approximately 2) is very similar to the EPR signals of compound I of ascorbate peroxidase and catalase from Micrococcus lysodeikticus, in which the radical has been identified as a porphyrin pi-cation radical. On the basis of the analysis of our data and comparison with the earlier published results for compounds I of other peroxidases, we interpret the LiP compound I signal by a model for exchange coupling between an S = 1 oxyferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S = 1/2) [Schulz, C.E., et al. (1979) FEBS Lett. 103, 102-105]. The exchange coupling is characterized by ferromagnetic rather than an antiferromagnetic interaction between the two species. The ferric-Lip EPR signal suggests that the iron in the heme is in near perfect orthogonal symmetry and provides additional evidence of the ferromagnetic interaction between the oxoferryl iron center and the porphyrin pi-cation radical.

Basidiomycota↗
PubMed1997

Spectral changes of lignin peroxidase during reversible inactivation.

The heme environment of lignin peroxidase (LiP) has been investigated by electronic absorption and electron paramagnetic resonance (EPR) spectroscopy. Native LiP was a pentacoordinate, high-spin ferric iron with a high-spin absorption band at 634 nm and g values at 5.86 and 2.07 in the EPR spectrum. Upon thermal inactivation, calcium ions were released from the enzyme and the Soret absorption decreased and red-shifted about 2 nm, the high-spin absorption band at 634 nm disappeared, and a low-spin absorption band appeared at 532 nm. The EPR spectrum and the temperature dependence of electronic absorption spectra revealed that the heme iron of the thermally inactivated enzyme was a mixture of high- and low-spin states, which was further supported by the changes in the electronic absorption and EPR spectra when cyanide was added to the thermally inactivated enzyme. Addition of various imidazoles or CN- to thermally inactivated enzyme demonstrated that the low-spin heme iron of inactivated enzyme was hexacoordinate with a distal histidine as its sixth ligand, in contrast to the active enzyme, which was pentacoordinate and high-spin. Upon addition of calcium to recover the thermally inactivated LiP, the reactivated enzyme had absorptions at 408, 502, and 634 nm and g values at 5.86 and 2.07 in the EPR spectrum, which demonstrated that the heme iron of the reactivated enzyme was again high-spin and pentacoordinated.

Calcium↗