The One-Electron Reduction Potential of 3-Amino-1, 2, 4-benzotriazine 1, 4-dioxide (Tirapazamine): A Hypoxia-Selective Bioreductive Drug

The one-electron reduction potential of 3-amino-l, 2, 4-benzotriazine 1, 4-dioxide, tirapazamine (SR 4233) in aqueous solution has been determined by pulse radiol-ysis. Reversible electron transfer was achieved between radiolytically-generated one-electron reduced radicals of tirapazamine (T), and quinones or benzyl viologen as redox standards. The reduction potential E_m7(T/T^±) was -0.45 ± 0.01 V vs. NHE at pH 7. From the pH dependence of the reduction potential, pK_a = 5.6 ± 0.2 was estimated for the tirapazamine radical, a value similar to the pK_a determined by other methods.     

The role of histidine-42 in the oxidation-reduction mechanism of Chromatium vinosum high potential iron-sulfur protein

The second order rate constants for the oxidation of high potential iron-sulfur protein (Hipip) of Chromatium vinosum by ferricyanide were determined as a function of ionic strength and pH. From the ionic strength results, calculations were done to correct the rate constant at each pH for the electrostatic interactions between Hipip and ferricyanide. The electrostatic corrections are necessary since the charge of the protein changes as a function of pH and can mask the ionization of mechanistically important amino acid residues. An apparent pK_a 7 was obtained from electrostatically corrected rate-pH profile, indicating the possible participation of histidine. Perturbation difference spectroscopic studies of Hipip as a function of pH also gave apparent pK_a values of 6.9 and 6.7 for the reduced and oxidized protein, respectively. That it was indeed His 42 (the only His in the polypeptide) that was responsible for the kinetic and spectroscopic pK_a values was demonstrated by modification of His 42 of Hipip by the histidine selective reagent diethylpyrocarbonate. No modification of Tyr 19 could be detected. It is concluded that either deprotonation or modification of His 42 results in the destabilization of the reduced cluster and thus a faster rate of oxidation. This work provides the first experimental evidence of the ‘squeeze effect’ mechanism (Carter, C.W., Jr., Kraut, J., Freer, S.T. and Alden, R.A. (1974) J. Biol. Chem. 249, 6339–6346) in which the polypeptide directly modulates the stability of the iron-sulfur cluster.     

NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase from Phanerochaete chrysosporium. Comparison with horseradish peroxidase

One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting state and the low spin, cyanide-inhibited derivative of isozyme H2 of the lignin peroxidase, LiP, from Phanerochaete chrysosporium strain BKM 1767. One-dimensional NMR revealed a resting state LiP that is five coordinate at 25 degrees C with an electronic structure similar to that of horseradish peroxidase, HRP. Differential paramagnetic relaxivity was used to identify the C beta H signals of the axial His177. A combination of bond correlation spectroscopy and nuclear Overhauser effect spectroscopy of cyanide-inhibited LiP (LiP-CN) has allowed the assignment of all resolved heme resonances without recourse to isotope labeling, as well as those of the proximal His177 and the distal His48. The surprising effectiveness of the two dimensional NMR methods on such a large and paramagnetic protein indicates that such two dimensional experiments can be expected to have major impact on solution structure determination of diverse classes of heme peroxidases. The two dimensional NMR data of LiP-CN reveal a heme contact shift pattern that reflects a close similarity to that of HRP-CN, including the unusual in-plane trans and cis orientation of the 2- and 4-vinyls. The axial His177 also exhibits the same orientation relative to the heme as in HRP-CN. The proximal His177 contact shifted resonances of both the low spin LiP-CN and high spin LiP are shown to reflect significantly reduced hydrogen bond donation by, or imidazolate character for, the axial histidine in LiP relative to HRP, which may explain the higher redox potential of LiP. The signals are identified for a distal residue that originates from the protonated His48 with disposition relative to the heme similar to that found for the distal His42 in HRP-CN. In contrast, the absence of any resolved signals attributable to an Arg44 in LiP-CN suggest that this distal residue has an altered orientation relative to the heme compared with that of the conserved Arg38 in HRP-CN (Thanabal, V., de Ropp, J. S., and La Mar, G. N. (1987) J. Am. Chem. Soc. 109, 7516-7525).     

Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are structurally similar heme-containing enzymes secreted by white-rot fungi. Unlike MnP, which is only specific for Mn(2+), LiP has broad substrate specificity, but it is not known if this versatility is due to multiple substrate-binding sites. We report here that a S168W variant of MnP from Phanerochaete chrysosporium not only retained full Mn(2+) oxidase activity, but also, unlike native or recombinant MnP, oxidized a multitude of LiP substrates, including small molecule and polymeric substrates. The kinetics of oxidation of most nonpolymeric substrates by the MnP variant and LiP were similar. The stoichiometries for veratryl alcohol oxidation by these two enzymes were identical. Some readily oxidizable substrates, such as guaiacol and ferrocyanide, were oxidized by MnP S168W and LiP both specifically and nonspecifically while recombinant MnP oxidized these substrates only nonspecifically. The functional similarities between this MnP variant and LiP provide evidence for the broad substrate specificity of a single oxidation site near the surface tryptophan.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

Lignin and manganese peroxidase activity in extracts from straw solid substrate fermentations

Manganese and lignin peroxidase (MnP, LiP) activities were measured in straw extracts from cultures of Phanerochaete chrysosporium. Out of six MnP substrates, the MBTH/DMAB (3-methyl-2-benzothiazolinone hydrazone/3-(dimethylamino)benzoic acid), gave the highest MnP activity. Detection of LiP activity as veratryl alcohol oxidation was inhibited by phenols in the straw culture extracts. Appropriate levels of veratryl alcohol and peroxide (4 mM and 0.4 mM, respectively), and a restricted sample volume (not larger than 10%) were necessary to detect activity.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms

Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity.     

黄孢原毛平革菌木素过氧化物酶类在天然木素降解中作用的研究

通过诱变得到十一株木素过氧化物酶酶活降低的黄孢原毛平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ）突变株,用灰色理论分析了其木素过氧化物酶类的产生与木素降解能力间的相关性,并从中筛选到一株木素过氧化物酶缺陷、锰过氧化物酶酶活明显降低的突变株,其木素降解能力为原始菌株的８０％左右。该菌粗酶液作用于纤维素酶酶解杉木木素和天然褐腐木素,可产生小分子的木素降解产物,此反应不需Ｈ２Ｏ２参与。红外光谱分析表明粗酶液对木素的作用主要为氧化作用,因此推测此突变株粗酶液中含有不同于木素过氧化物酶和锰过氧化物酶的与木素氧化降解有关的酶类     

Degradation of 4-chlorophenol by the white rot fungus Phanerochaete chrysosporium in free and immobilized cultures

4-Chlorophenol (4-CP) degradation was investigated by suspended and immobilized Phanerochaete chrysosporium conducted in static and agitated cultures. The best results were achieved when experiment was carried out in a rotating biological contactor instead of an Erlenmeyer flask, for both batch degradation and repeated batch degradation. The relative contribution of lignin peroxidase (LiP) versus manganese peroxidase (MnP) to the 4-CP degradation by P. chrysosporium was investigated. 4-CP degradation slightly increased and a high level of MnP (38 nKat ml(-1)) was produced when P. chrysosporium was grown at high Mnll concentration. High LiP production in the medium had no significant effect on 4-CP degradation. 4-CP degradation occurred when P. chrysosporium was grown in a medium that repressed LiP and MnP production. This result indicates that LiP and MnP are not directly involved in 4-CP degradation by P. chrysosporium.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Optimum stability conditions of pH and temperature for ligninase and manganese-dependent peroxidase from Phanerochaete chrysosporium. Application to in vitro decolorization of Poly R-478 by MnP

Summary In the present work, the two main factors affecting enzymatic stability, i.e. pH and temperature, were analysed in order to determine the optimum ones to maintain ligninase (LiP) and manganese-dependent peroxidase (MnP) activities for prolonged periods of time. The optimum pH and temperature range obtained was around 4.2 and 34 °C for the former and 4.5 and 32 °C for the latter. Under these conditions LiP and MnP showed a half-life time of about 100 and 500 h, respectively. In addition, extracellular liquid containing mainly MnP (200 U/l) was able to decolorize about 20% of the polymeric dye Poly R-478 in 15 min. The decolorization was carried out at a pH of 4.5 (6 mM sodium malonate) and a temperature of 30 °C.     

Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site

The three-dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu-35, Glu-39, and Asp-179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley-Liss, Inc.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to LiP of Phanerochaete chrysosporium and a total inhibition of LiP was observed.     

Solid state fermentation of Achras zapota lignocellulose by Phanerochaete chrysosporium

The biological transformation of lignocellulose of Achras zapota by white rot fungi, Phanerochaete chrysosporium, in solid state fermentation (SSF) was studied for 28 days. The kinetic transformation of lignocellulose was monitored through the determination of acid soluble and acid insoluble lignin content, total organic carbon (TOC) and chemical oxygen demand (COD). The lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP) were quantified on weekly intervals. The degradation of lignin and other structural moieties of A. zapota lignocellulose were confirmed by high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The degradation of lignin was increased after 7 days of fermentation with the release of water soluble and fermentable products. The LiP and MnP activities were increased in the first week of SSF and lignin degradation was also set to increase. This was accompanied with increase in COD by 94.6% and TOC by 80% and lignin content was decreased by 76%. The maximum activities of the enzymes LiP and MnP in extracellular fluid of SSF under nitrogen limitation, at pH 5.0, at temperature 37 degrees C and at 60% humidity were 2100 U/L and 1200 U/L.     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.     

Homology modelling and H NMR studies of human leukaemia inhibitory factor

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with ¹H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pK_a values of which range from 3.6 to 7.4. These pK_a values were assigned to individual histidine residues from NMR studies on a series of His → Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.     

Study of the swelling dynamics with overshooting effect of hydrogels based on sodium alginate-g-acrylic acid

A series of hydrogels were synthesized by graft cross-link copolymerization of sodium alginate (SA) and acrylic acid (AA) using N, N-methylene-bis-(acrylamide) as a cross-linker. By study of the swelling kinetics of the hydrogels in different buffer solutions, the overshooting effect was observed in acidic medium, namely the gels firstly swelled to a maximum value following by a gradual deswelling until the equilibrium. The phenomenon is interpreted as a cooperative physical cross-linking caused by the hydrogen bond formation between the carboxyl groups of the hydrogels in a hydrophobic environment. The hydrogen bond formation was further confirmed by FT-IR spectra. The dependence of overshooting effect on the pH of buffer solution was more noticeable in comparison with the composition of hydrogels, demonstrating that the cooperative physical cross-linking caused by the hydrogen bond formation is dominant. Whether or not the overshooting effect appears is not only relative to the pH of buffer solution, but also depends on the pK_a of carboxyl groups on the network. The overshoot processes of the hydrogels under acidic medium at pH below the pK_a follow a quantitative model proposed by Díez-Peńa et al., and the theoretical curves are in very good agreement with the experimental data. While in pH > pK_a buffer solutions, the overshoot phenomenon does not appear arising from the repulsive interaction between the ionized carboxyl groups, the swelling processes follow Schott second-order rate equation.     

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.