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Yarman A, Gröbe G, Neumann B, Kinne M, Gajovic-Eichelmann N, Wollenberger U, Hofrichter M, Ullrich R, Scheibner K, Scheller FW (2012). "The aromatic peroxygenase from Marasmius rutola--a new enzyme for biosensor applications". Anal. Bioanal. Chem., 402: 405-412.
The aromatic peroxygenase (APO; EC 220.127.116.11) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe(2+)/Fe(3+) redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.
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Barková K, Kinne M, Ullrich R, Hennig L, Fuchs A, Hofrichter M (2011). "Regioselective hydroxylation of diverse flavonoids by an aromatic peroxygenase". Tetrahedron, 67: 4874-4878.
Aromatic peroxygenases are extracellular fungal biocatalysts that selectively oxidize a variety of organic compounds. We found that the peroxygenase of the fungus Agrocybe aegerita
APO) catalyzes the H2
-dependent hydroxylation of diverse flavonoids. The reactions proceeded rapidly and regioselectively yielding preferentially monohydroxylated products, e.g., from flavanone
as well as daidzein
, and genistein
. In addition to hydroxylation, O
-demethylation of fully methoxylated tangeretin
was catalyzed by Aae
APO. The enzyme was merely lacking activity on the quercetin
, maybe due to sterical hindrance by the bulky sugar substituents. Mechanistic studies indicated the presence of epoxide intermediates during hydroxylation and incorporation of H2
into the reaction products. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of flavonoid metabolites.
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Bernini C, Pogni R, Ruiz-Dueñas FJ, Martínez AT, Basosi R, Sinicropi A (2011). "EPR parameters of amino acid radicals in P. eryngii versatile peroxidase and its W164Y variant computed at the QM/MM level". Phys. Chem. Chem. Phys., 13: 5078-5098.
Quantum mechanics/molecular mechanics (QM/MM) methods, employing density functional theory (DFT), have been used to compute the electron paramagnetic resonance (EPR) parameters of tryptophan and tyrosyl radical intermediates involved in the catalytic cycle of Pleurotus eryngii versatile peroxidase (VP) and its W164Y variant, respectively. These radicals have been previously experimentally detected and characterized both in the two-electron and one-electron activated forms of the enzymes. In this work, the well-studied W164 radical in VP has been chosen for calibration purposes because its spectroscopic properties have been extensively studied by multifrequency EPR and ENDOR spectroscopies. Using a B3LYP/CHARMM procedure, appropriately accounting for electrostatic, such as hydrogen bonding, and steric environmental interactions, a good agreement between the calculated and measured EPR parameters for both radicals has been achieved; g-tensors, hyperfine coupling constants (hfcc) and Mulliken spin densities have been correlated to changes in geometries, hydrogen bond networks and electrostatic environment, with the aim of understanding the influence of the protein surroundings on EPR properties. In addition, the present calculations demonstrate, for VP, the formation of a neutral tryptophan radical, hydrogen bonded to the nearby E243, via a stepwise electron and proton transfer with earlier involvement of a short-lived tryptophan cationic species. Instead, for W164Y, the QM/MM dynamics simulation shows that the tyrosine oxidation proceeds via a concerted electron and proton transfer and is accompanied by a significant reorganization of residues and water molecules surrounding the tyrosyl radical.
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Gröbe G, Ullrich R, Pecyna MJ, Kapturska D, Friedrich S, Hofrichter M, Scheibner K (2011). "High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula". AMB Express, 1: 31-42.
An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities.
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Gutiérrez A, Babot ED, Ullrich R, Hofrichter M, Martínez AT, del Río JC (2011). "Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase". Arch. Biochem. Biophys., 541: 33-43.
Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C(12)-C(20) saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H(2)(18)O(2) established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates.
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Hernández-Ortega A, Borrelli K, Ferreira P, Medina M, Martínez AT, Guallar V (2011). "Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase". Biochem. J., 431: 341-350.
Aryl-alcohol oxidase (AAO) provides H2O2 in fungal degradation of lignin, a process of high biotechnological interest. Its crystal structure does not show an open access to the active site, where different aromatic alcohols are oxidized. We studied substrate diffusion and oxidation in AAO, compared with the structurally-related choline oxidase (CHO). Cavity finder and ligand diffusion simulations indicate the substrate entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr92. Mixed quantum mechanics/molecular mechanics (QM/MM) studies combined with site-directed mutagenesis showed two active-site catalytic histidines, whose substitution strongly decreased both catalytic and (transient-state) reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis, and pH profiles give a robust answer about the nature of the catalytic base. The histidine in front of the FAD ring, AAO His502 (and CHO His466), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO.
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Hernández-Ortega A, Lucas F, Ferreira P, Medina M, Guallar V, Martínez AT (2011). "Modulating O2 reactivity in a fungal flavoenzyme: Involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine". J. Biol. Chem., 286: 41105-41114.
Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O2 to H2O2 in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations showed O2 access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O2 reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substituting Phe-501 by tryptophan. The high oxygen catalytic efficiency of F501W, ~2 fold that of native AAO and ~120 fold that of F501A, seems related to a higher O2 availability since the turnover number was slightly decreased with respect to native enzyme. Free-diffusion simulations of O2 inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded over 60% O2 population at 3-4 A from the flavin C4a in F501W, compared with 44% in AAO and only 14% in F501A. Paradoxically, the O2 reactivity of AAO decreases when the access channel is enlarged, and increases when it is constricted by introducing a tryptophan residue. This is because the side chain of Phe501, contiguous to the catalytic histidine (His-502 in AAO), helps O2 positioning at adequate distance from the flavin C4a (and His-502 Nε). Phe-501 substitution by a bulkier tryptophan residue enabled an increase in the O2 reactivity of this flavoenzyme.
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Kinne M, Poraj-Kobielska M, Ullrich R, Nousiainen P, Sipilä J, Scheibner K, Hammel KE, Hofrichter M (2011). "Oxidative cleavage of non-phenolic β-O-4 lignin model dimers by an extracellular aromatic peroxygenase". Holzforschung, doi: 10.1515/HF.2011.057.
The extracellular aromatic peroxygenase of the agaric fungus Agrocybe aegerita
catalyzed the H2
-dependent cleavage of non-phenolic arylglycerol-β
-aryl ethers (β
-O-4 ethers). For instance 1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)propane-1,3-diol, a recalcitrant dimeric lignin model compound that represents the major non-phenolic substructure in lignin, was selectively O
-demethylated at the para
-methoxy group to give formaldehyde and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol. The phenol moiety of the latter compound was then enzymatically oxidized into phenoxy radicals and a quinoid cation, which initiated the autocatalytic cleavage of the dimer and the formation of monomers such as 2-methoxy-1,4-benzoquinone and phenoxyl-substituted propionic acid. The introduction of 18
O from H218
O at different positions into the products provided information about the routes of ether cleavage. Studies with a 14
C-labeled lignin model dimer showed that more than 70% of the intermediates formed were further coupled to form polymers with molecular masses above 10 kDa. The results indicate that fungal aromatic peroxygenases may be involved in the bioconversion of methoxylated plant ingredients originating from lignin or other sources.
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Liers C, Arnstadt T, Ullrich R, Hofrichter M (2011). "Patterns of lignin degradation and oxidative enzyme secretion by different wood- and litter-colonizing basidiomycetes and ascomycetes grown on beech wood". FEMS Microbiol. Ecol., 78: 91-102.
The degradation of lignocellulose and the secretion of extracellular oxidoreductases were investigated in beech-wood (Fagus sylvatica
) microcosms using 11 representative fungi of four different eco-physiological and taxonomic groups causing: i) classic white-rot of wood (e.g. Phlebia radiata
), ii) “non-specific” wood-rot (e.g. Agrocybe aegerita
), iii) white-rot of leaf-litter (Stropharia rugosoannulata
) or iv) soft-rot of wood (e.g. Xylaria polymorpha
). All strong white-rotters produced manganese-oxidizing peroxidases as the key enzymes of ligninolysis (75–2200 mU g−1
), while activity of lignin peroxidase was not detectable in the wood extracts. Interestingly, activities of two just recently discovered peroxidases, namely aromatic peroxygenase and a manganese-independent peroxidase of the DyP-type were detected in the culture extracts of A. aegerita
(up to 125 mU g−1
) and Auricularia auricula-judae
(up to 400 mU g−1
), respectively. The activity of classic peroxidases correlated to some extent with the removal of wood components (e.g. Klason lignin) and the release of small water-soluble fragments (0.5–1.0 kDa) characterized by aromatic constituents, while the laccase activity rather correlated with the formation of high-molecular mass fragments (30–200 kDa). The differences observed in the degradation patterns allow to distinguish between the rot-types caused by basidio- and ascomycetes, and may be suitable for following the effects of oxidative key enzymes (ligninolytic peroxidases vs
. laccases, role of novel peroxidases) during wood decay.
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Liers C, Ullrich R, Hofrichter M, Minibayeva F, Beckett RP (2011). "A heme peroxidase of the ascomyceteous lichen Leptogium saturninum oxidizes high-redox potential substrates". Fungal Gen. Biol., 48: 1139-11.
Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosinase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum (LsaPOX) by fast protein liquid chromatography and then partially characterized.
The oligomeric protein occurs as both 79 kDa dimeric and 42 kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6-dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auricula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fischeri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant peroxidases.
POX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow
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Marques G, Molina S, Babot ED, Lund H, del Río JC, Gutiérrez A (2011). "Exploring the potential of fungal manganese-containing lipoxygenase for pitch control and pulp delignification". Bioresource Technol., 102: 1338-1343.
The potential of the lipoxygenase from Gaeumannomyces graminis to remove lipophilic extractives from eucalypt and flax pulps was investigated. Pulp treatments were performed with the lipoxygenase both in the presence and absence of linoleic acid, and were followed by a peroxide bleaching stage. The main lipophilic extractives from eucalypt pulp such as conjugated and free sterols decreased up to 40% and 7%, respectively, by the lipoxygenase treatment in the presence of linoleic acid. Different degradation patterns were observed among the lipophilic compounds present in flax pulp, although a high removal of all the extractives classes, including alkanes (21-55%), fatty alcohols (42-61%), and free (16-55%) and glycosylated (45-71%) sterols, was attained in all the lipoxygenase treatments. Reactions of the lipoxygenase with model lipid mixtures were carried out to better understand the degradation patterns observed in pulps. Finally, pulp delignification by the lipoxygenase treatments was also evaluated.
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Martínez AT, Rencoret J, Nieto L, Jiménez-Barbero J, Gutiérrez A, del Río JC (2011). "Selective lignin and polysaccharide removal in natural fungal decay of wood as evidenced by in situ structural analyses". Environ. Microbiol., 13: 96-107.
Selective modification/degradation of the main plant polymers (cellulose, hemicelluloses and lignin) was investigated in a hardwood after white and brown-rot fungal decay under environmental conditions. The chemical changes produced in the plant cell wall were analysed in situ, by nuclear magnetic resonance (NMR) at the gel state, and analytical pyrolysis. Two-dimensional (2D) NMR of the white-rotted wood showed only cellulose and (deacetylated) hemicellulose, and the complete removal of lignin. On the other hand, the brown-rotted wood showed the nearly complete absence of polysaccharides, while the main features of lignin structure, as revealed by 2D-NMR, could be observed. These included well-resolved aromatic and side-chain cross-signals, although the intensity of the latter signals was lowered indicating a reduction in the number of side-chain linkages (beta-O-4\' and beta-beta\') per aromatic unit (their relative abundances remaining unchanged). These results contrast with a recent study concluding that the aromatic polymer after brown-rot decay is not longer recognized as lignin. Some oxidative alteration of lignin during brown-rot decay was evidenced and, more interesting, several compounds with 3-methoxycatechol skeleton were released upon pyrolysis. Lignin demethylation is consistent with recent brown-rot transcriptomic/secretomic studies showing overexpression of methanol oxidase, which could use lignin-derived methanol to generate the peroxide required for cellulose depolymerization via Fenton chemistry.
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Miki Y, Calviño FR, Pogni R, Giansanti S, Ruiz-Dueñas FJ, Martínez MJ, Basosi R, Romero A, Martínez AT (2011). "Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine". J. Biol. Chem., 286: 15525-15534.
Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiP and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques.
The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146 and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome-c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by one electron abstraction with formation of substrate cation radical, as described for the well-known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low-temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical.
The crystal structure reveals a partially-exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: i) enabling substrate recognition by aromatic interactions; ii) as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and iii) providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.
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Peter S, Kinne M, Wang X, Ullrich R, Kayser G, Groves JT, Hofrichter M (2011). "Selective hydroxylation of alkanes by an extracellular fungal peroxygenase". FEBS J., 278: 3667-3675.
Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H2
as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita
catalyzes the H2
-dependent hydroxylation of linear alkanes at the 2-position and 3-position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n
-heptane and n
-octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R
)-enantiomer of the corresponding 3-alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H218
resulted in complete incorporation of 18
O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n
showed a large intramolecular deuterium isotope effect [(kH
] of 16.0 ± 1.0 for 2-hexanol and 8.9 ± 0.9 for 3-hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 1011
. These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long-chain alkanes (> C16
) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood.
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Rencoret J, Gutiérrez A, Nieto L, Jiménez-Barbero J, Faulds CB, Kim H, Ralph J, Martínez AT, del Río JC (2011). "Lignin composition and structure in young versus adult Eucalyptus globulus plants". Plant Physiol., 155: 667-682.
Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied “in situ” by a new procedure enabling the acquisition of 2D-NMR spectra on wood gels formed in the NMR tube, as well as by analytical pyrolysis (Py-GC/MS). In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, Py-GC/MS, and thioacidolysis. The data indicated that phydroxyphenyl (H) and guaiacyl (G) units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that β-O-4′ and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open β-1′ structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end-groups were epleted with age, while spirodienone abundance increased, and the main substructures (β-O-4′ and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important non-ether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed G-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels, without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are ~50% acetylated) and 4-O-methylglucuronosyl signals.
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Ruiz-Dueñas FJ, Fernandez-Fueyo E, Martínez MJ, Martínez AT (2011). "Pleurotus ostreatus heme peroxidases: An in silico analysis from the genome sequence to the enzyme molecular structure". C. R. Biologies, 334: 795-805.
An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal peroxidases revealed the complete inventory of heme peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the ‘‘classical’’ superfamily of plant, fungal, and bacterial peroxidases; and members of two relatively ‘‘new’’ peroxidase superfamilies, namely heme-thiolate peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing peroxidases, already known in P. ostreatus but still to be thoroughly explored and characterized.
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Aranda E, Ullrich R, Hofrichter M (2010). "Conversion of polycyclic aromatic hydrocarbons, methyl naphthalenes and dibenzofuran by two fungal peroxygenases". Biodegradation, 21: 267-281.
The aim of this work has been to study the substrate specificity of two aromatic peroxygenases concerning polyaromatic compounds of different size and structure as well as to identify the key metabolites of their oxidation. Thus, we report here on new pathways and reactions for 2-methylnaphthalene, 1-methylnaphthalene, dibenzofuran, fluorene, phenanthrene, anthracene and pyrene catalyzed by peroxygenases from Agrocybe aegerita and Coprinellus radians (abbreviated as AaP and CrP). AaP hydroxylated the aromatic rings of all substrates tested at different positions, whereas CrP showed a limited capacity for aromatic ring-hydroxylation and did not hydroxylate phenanthrene but preferably oxygenated fluorene at the non-aromatic C(9)-carbon and methylnaphthalenes at the side chain. The results demonstrate for the first time the broad substrate specificity of fungal peroxygenases for polyaromatic compounds, and they are discussed in terms of their biocatalytic and environmental implications.
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Ferreira P, Hernández-Ortega A, Herguedas B, Rencoret J, Gutiérrez A, Martínez MJ, Jiménez-Barbero J, Medina M, Martínez AT (2010). "Kinetic and chemical characterization of aldehyde oxidation by fungal aryl-alcohol oxidase". Biochem. J., 425: 585-593.
Fungal aryl-alcohol oxidase (AAO) provides H2O2 for lignin biodegradation. AAO is active on benzyl alcohols that are oxidized to aldehydes. However, the H2O2 formed from some of them was more than stoichiometric with respect to the aldehyde detected. This was due to a double reaction that involves aryl-aldehyde oxidase activity. The latter was investigated using different benzylic aldehydes, whose oxidation to acids was demonstrated by GC-MS. The steady and pre-steady state kinetic constants together with the chromatographic results revealed a strong influence of substrate electron withdrawing/donating substituents on activity, being the highest on p-nitrobenzaldehyde and halogenated aldehydes and the lowest on methoxylated aldehydes. Moreover, activity was correlated to the aldehyde hydration rates estimated by 1H-NMR. These findings, together with the absence in the AAO active site of a residue able to drive oxidation via an aldehyde thiohemiacetal, suggested that oxidation mainly proceeds via the gem-diol species. The reaction mechanism (with solvent isotope effect of D2Okred ~1.5) would be analogous to that described for alcohols, the reductive half-reaction involving concerted hydride transfer from α-carbon, and proton abstraction from one of the gem-diol hydroxyls by a base. The existence of two steps of opposite polar requirements (hydration and hydride transfer) explains some aspects of aldehyde oxidation by AAO. Site-directed mutagenesis identified two histidines strongly involved in gem-diol oxidation and, unexpectedly, suggested that an active-site tyrosine could facilitate the oxidation of some aldehydes showing no detectable hydration. Double alcohol and aldehyde oxidase activities of AAO would contribute to H2O2 supply by the enzyme.
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García-Ruiz E, Maté D, Ballesteros A, Martínez AT, Alcalde M (2010). "Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast". Microb. Cell Fact., 9: 17-29.
BACKGROUND: In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae. RESULTS: Taking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues. CONCLUSIONS: The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.
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Gutiérrez A, del Río JC, Martínez AT (2010). "Fungi and Their Enzymes for Pitch Control in the Pulp and Paper Industry". The Mycota, 10: 357-377.
Pitch biocontrol was the first example where microbial biotechnology provided successful solutions to the pulp and paper sector. Triglycerides cause deposits in mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi, and the latter are improved lipases. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the selectivity of the laccase-mediator system permits its integration as an additional bleaching stage providing a double benefit: pitch biocontrol and removal of residual lignin in chlorine-free pulp bleaching.
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