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Hofrichter M, Ullrich R (2010). "New Trends in Fungal Biooxidation". The Mycota, 10: 425-449.
Biooxidations using isolated biocatalysts for the oxidative conversion of organic compounds and materials are becoming more and more important in the industrial sector. Research in this direction is part of the rapidly developing field of White Biotechnology and is carried out not least against the background of sustainable development and the need to replace environmentally risky technologies by eco-friendly processes. Whereas whole cells are already widely used as oxidative biocatalysts, the application of isolated enzymes is still limited to a few examples. One reason for that is the cost-intensive production and laborious purification of enzymes which are mostly intracellular and sometimes membrane-bound proteins with low stability and complex co-factor requirements. The use of secreted oxidoreductases offers several advantages: they are easier to separate and purify, need only cheap co-substrates such as dioxygen or peroxides and are far more stable than intracellular enzymes. Filamentous fungi secrete a broad spectrum of oxidative biocatalysts which are involved, amongst others, in the degradation of recalcitrant biopolymers, the synthesis of melanins as well as the detoxification of plant ingredients, microbial metabolites and organopollutants. We focus here on novel secreted enzymes - peroxygenases and DyP-type peroxidases - with remarkable catalytic properties. Furthermore selected features of “classic” fungal oxidoreductases, such as laccase, tyrosinase and chloroperoxidase, are discussed against the background of innovative recent developments in the field of enzyme application.
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Hofrichter M, Ullrich R, Pecyna MJ, Liers C, Lundell T (2010). "New and classic families of secreted fungal heme peroxidases". Appl. Microbiol. Biotechnol., 87: 871-897.
Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the "classic" fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.
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Kinne M, Zeisig C, Ullrich R, Kayser G, Hammel KE, Hofrichter M (2010). "Stepwise oxygenations of toluene and 4-nitrotoluene by a fungal peroxygenase". Biochem. Biophys. Res. Commun., 397: 18-21.
Fungal peroxygenases have recently been shown to catalyze remarkable oxidation reactions. The present study addresses the mechanism of benzylic oxygenations catalyzed by the extracellular peroxygenase of the agaric basidiomycete Agrocybe aegerita . The peroxygenase oxidized toluene and 4-nitrotoluene via the corresponding alcohols and aldehydes to give benzoic acids. The reactions proceeded stepwise with total conversions of 93% for toluene and 12% for 4-nitrotoluene. Using H 2 18 O 2 as the co-substrate, we show here that H 2 O 2 is the source of the oxygen introduced at each reaction step. Agrocybe aegerita peroxygenase resembles cytochromes P450 and heme chloroperoxidase in catalyzing benzylic hydroxylations.
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Liers C, Bobeth C, Pecyna MJ, Ullrich R, Hofrichter M (2010). "DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes". Appl. Microbiol. Biotechnol., 85: 1869-1879.
The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l−1). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l−1. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn2+) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.
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Marques G, Gamelas JA, Ruiz-Dueñas FJ, del Río JC, Evtuguin DV, Martínez AT, Gutiérrez A (2010). "Delignification of eucalypt kraft pulp with manganese-substituted polyoxometalate assisted by fungal versatile peroxidase". Bioresource Technol., 101: 5935-5940.
Oxidation of the manganese-substituted polyoxometalate [SiW(11)Mn(II)(H(2)O)O(39)](6-) (SiW(11)Mn(II)) to [SiW(11)Mn(III)(H(2)O)O(39)](5-) (SiW(11)Mn(III)), one of the most selective polyoxometalates for the kraft pulp delignification, by versatile peroxidase (VP) was studied. First, SiW(11)Mn(II) was demonstrated to be quickly oxidized by VP at room temperature in the presence of H(2)O(2) (K(m)=6.4+/-0.7 mM and k(cat)=47+/-2s(-1)). Second, the filtrate from eucalypt pulp delignification containing reduced polyoxometalate was treated with VP/H(2)O(2), and 95-100% reoxidation was attained. In this way, it was possible to reuse the liquor from a first SiW(11)Mn(III) stage for further delignification, in a sequence constituted by two polyoxometalate stages, and a short intermediate step consisting of the addition of VP/H(2)O(2) to the filtrate for SiW(11)Mn(II) reoxidation. When the first ClO(2) stage of a conventional bleaching sequence was substituted by the two-stage delignification with polyoxometalate (assisted by VP) a 50% saving in ClO(2) was obtained for similar mechanical strength of the final pulp.
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Piontek K, Ullrich R, Liers C, Diederichs K, Plattner D, Hofrichter M (2010). "Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron". Acta Crystallogr.F Struct. Biol. Crystal. Commun., 66: 693-698.
Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H2O2 for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 Å resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 Å resolution by molecular replacement.
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Ruiz-Dueñas FJ, Martínez AT (2010). "Structural and Functional Features of Peroxidases with a Potential as Industrial Biocatalysts". Biocatalysts Based on Heme Peroxidases, E. Torres and M. Ayala (eds.), 3: 37-59.
This chapter begins with a description of the main structural features of heme peroxidases representative of the two large superfamilies of plant–fungal–bacterial and animal peroxidases, and the four additional (super)families described to date. Then, we focus on several fungal peroxidases of high biotechnological potential as industrial biocatalysts. These include (1) ligninolytic peroxidases from white-rot basidiomycetes being able to oxidize high redox-potential substrates at an exposed protein radical; (2) heme-thiolate peroxidases that are structural hybrids of typical peroxidases and cytochrome P450 enzymes and, after their discovery in sooty molds, are being described in basidiomycetes with even more interesting catalytic properties, such as selective aromatic oxygenation; and (3) the so-called dye-decolorizing peroxidases that are still to be thoroughly investigated but have been identified in different basidiomycete genomes. The structural–functional description of these peroxidases includes an analysis of the heme environment and a description of their substrate oxidation sites, with the purpose of understanding their interesting catalytic properties and biotechnological potential.
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Ferreira P, Hernández-Ortega A, Herguedas B, Martínez AT, Medina M (2009). "Aryl-alcohol oxidase involved in lignin degradation: A mechanistic study based on steady and pre-steady state kinetics and primary and solvent isotope effects with two alcohol substrates". J. Biol. Chem., 284: 24840-24847.
Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O2 reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and pre-steady state conditions (the latter showing ∼9-fold slower enzyme reduction when α-bideuterated substrates were used, and ∼13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate α-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide.
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Hofrichter M, Ullrich R, Pecyna MJ, Kinne M, Kluge M, Alcalde M, Liers C, Poraj-Kobielska M, Gröbe G, Scheibner K, Bittner B, Schubert R, Hammel KE (2009). "Aromatic peroxygenases from mushrooms: extracellular heme-thiolate proteins of a new enzyme sub-subclass?". Proc. 16th I.C. Cytochrome P450, H. Shoun and H. Ohkawa (Eds.): 83-87.
Kinne M, Poraj-Kobielska M, Ralph J, Ullrich R, Hofrichter M, Hammel KE (2009). "Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase". J. Biol. Chem., 284: 29343-29349.
Many litter-decay fungi secrete heme-thiolate peroxygenases that oxidize various organic chemicals, but little is known about the role or mechanism of these enzymes. We found that the extracellular peroxygenase of Agrocybe aegerita
catalyzed the H2
-dependent cleavage of environmentally significant ethers, including methyl t
-butyl ether, tetrahydrofuran, and 1,4-dioxane. Experiments with tetrahydrofuran showed the reaction was a two-electron oxidation that generated one aldehyde group and one alcohol group, yielding the ring-opened product 4-hydroxybutanal. Investigations with several model substrates provided information about the route for ether cleavage: (a
) steady-state kinetics results with methyl 3,4-dimethoxybenzyl ether, which was oxidized to 3,4-dimethoxybenzaldehyde, gave parallel double reciprocal plots suggestive of a ping-pong mechanism (Km(peroxide)
, 1.99 ± 0.25 mm
, 1.43 ± 0.23 mm
, 720 ± 87 s−1
) the cleavage of methyl 4-nitrobenzyl ether in the presence of H218
resulted in incorporation of 18
O into the carbonyl group of the resulting 4-nitrobenzaldehyde, and (c
) the demethylation of 1-methoxy-4-trideuteromethoxybenzene showed an observed intramolecular deuterium isotope effect [(kH
] of 11.9 ± 0.4. These results suggest a hydrogen abstraction and oxygen rebound mechanism that oxidizes ethers to hemiacetals, which subsequently hydrolyze. The peroxygenase appeared to lack activity on macromolecular ethers, but otherwise exhibited a broad substrate range. It may accordingly have a role in the biodegradation of natural and anthropogenic low molecular weight ethers in soils and plant litter.
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Kluge M, Ullrich R, Dolge C, Scheibner K, Hofrichter M (2009). "Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H(2)O (2) and intermediary epoxidation". Appl. Microbiol. Biotechnol., 81: 1071-1076.
Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A (max) 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH <7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using (18)O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.
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Martínez AT, Ruiz-Dueñas FJ, Martínez MJ, del Río JC, Gutiérrez A (2009). "Enzymatic delignification of plant cell wall: from nature to mill". Curr. Opin. Biotechnol., 20: 348-357.
Lignin removal is a central issue in paper pulp manufacture, and production of other renewable chemicals, materials, and biofuels in future lignocellulose biorefineries. Biotechnology can contribute to more efficient and environmentally sound deconstruction of plant cell wall by providing tailor-made biocatalysts based on the oxidative enzymes responsible for lignin attack in Nature. With this purpose, the already-known ligninolytic oxidoreductases are being improved using (rational and random-based) protein engineering, and still unknown enzymes will be identified by the application of the different 'omics' technologies. Enzymatic delignification will be soon at the pulp mill (combined with pitch removal) and our understanding of the reactions produced will increase by using modern techniques for lignin analysis.
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Martínez D, .... , Ferreira P, Ruiz-Dueñas FJ, Martínez AT, .... , Cullen D (2009). "Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion". Proc. Natl. Acad. Sci. USA, 106: 1954-1959.
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β-1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.
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Miki Y, Morales M, Ruiz-Dueñas FJ, Martínez MJ, Wariishi H, Martínez AT (2009). "Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue". Protein Expr. Purif., 68: 208-214.
Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca2+ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H2O2 and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (kcat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (Km) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.
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Pecyna MJ, Ullrich R, Bittner B, Clemens A, Scheibner K, Schubert R, Hofrichter M (2009). "Molecular characterization of aromatic peroxygenase from Agrocybe aegerita". Appl. Microbiol. Biotechnol., 84: 885-897.
Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita
or Coprinellus radians
catalyze reactions—for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine—which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita
1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo
1 in A. aegerita
. The full-length sequences of A. aegerita
peroxygenase as well as a partial sequence of C. radians
peroxygenase confirmed the enzymes’ affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago
(and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO’s widespread occurrence in the fungal kingdom.
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Romero E, Ferreira P, Martínez AT, Martínez MJ (2009). "New oxidase from Bjerkandera arthroconidial anamorph that oxidizes both phenolic and nonphenolic benzyl alcohols". Biochim. Biophys. Acta, 1794: 689-697.
A new flavooxidase is described from a Bjerkandera arthroconidial anamorph. Its physicochemical characteristics, a monomeric enzyme containing non-covalently bound flavin adenine dinucleotide (FAD), and several catalytic properties, such as oxidation of aromatic and polyunsaturated aliphatic primary alcohols, are similar to those of Pleurotus eryngii aryl-alcohol oxidase (AAO). However, it also efficiently oxidizes phenolic benzyl and cinnamyl alcohols that are typical substrates of vanillyl-alcohol oxidase (VAO), a flavooxidase from a different family, characterized by its multimeric nature and presence of covalently-bound FAD. The enzyme also differs from P. eryngii AAO by having extremely high efficiency oxidizing chlorinated benzyl alcohols (1000-1500 s(-1) mM(-1)), a feature related to the different alcohol metabolites secreted by the Pleurotus and Bjerkandera species including chloroaromatics, and higher activity on aromatic aldehydes. What is even more intriguing is the fact that, the new oxidase is optimally active at pH 6.0 on both p-anisyl and vanillyl alcohols, suggesting a mechanism for phenolic benzyl alcohol oxidation that is different from that described in VAO, which proceeds via the substrate phenolate anion formed at basic pH. Based on the above properties, and its ADP-binding motif, partially detected after N-terminus sequencing, the new enzyme is classified as a member of the GMC (glucose-methanol-choline oxidase) oxidoreductase family oxidizing both AAO and VAO substrates.
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Ruiz-Dueñas FJ, Martínez AT (2009). "Microbial degradation of lignin: How a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this". Microbial Biotechnol., 2: 164-177.
Lignin is the second most abundant constituent of the cell wall of vascular plants, where it protects cellulose towards hydrolytic attack by saprophytic and pathogenic microbes. Its removal represents a key step for carbon recycling in land ecosystems, as well as a central issue for industrial utilization of plant biomass. The lignin polymer is highly recalcitrant towards chemical and biological degradation due to its molecular architecture, where different non-phenolic phenylpropanoid units form a complex three-dimensional network linked by a variety of ether and carbon–carbon bonds. Ligninolytic microbes have developed a unique strategy to handle lignin degradation based on unspecific one-electron oxidation of the benzenic rings in the different lignin substructures by extracellular haemperoxidases acting synergistically with peroxide-generating oxidases. These peroxidases posses two outstanding characteristics: (i) they have unusually high redox potential due to haem pocket architecture that enables oxidation of non-phenolic aromatic rings, and (ii) they are able to generate a protein oxidizer by electron transfer to the haem cofactor forming a catalytic tryptophanyl-free radical at the protein surface, where it can interact with the bulky lignin polymer. The structure–function information currently available is being used to build tailor-made peroxidases and other oxidoreductases as industrial biocatalysts.
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Ruiz-Dueñas FJ, Morales M, García-Ruiz E, Miki Y, Martínez MJ, Martínez AT (2009). "Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases". J. Exp. Bot., 60: 441-452.
Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn2+, the manganese peroxidase (MnP) substrate (Mn3+ being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these ‘hybrid’ properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn2+ oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not β-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.
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Ruiz-Dueñas FJ, Pogni R, Morales M, Giansanti S, Mate MJ, Romero A, Martínez MJ, Basosi R, Martínez AT (2009). "Protein radicals in fungal versatile peroxidase: Catalytic tryptophan radical in both Compound I and Compound II and studies on W164Y, W164H and W164S variants". J. Biol. Chem., 284: 7986-7994.
Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k2app) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k2) was more affected than the dissociation constant (KD2). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k3) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.
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Ullrich R, Liers C, Schimpke S, Hofrichter M (2009). "Purification of homogeneous forms of fungal peroxygenase". Biotechnol. J., 4: 1619-1626.
Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.
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