Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket.
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ABSTRACT:
SUBMITTER: Santucci R
PROVIDER: S-EPMC1221123 | biostudies-other | 2000 Jul
REPOSITORIES: biostudies-other
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Project description:The negative impacts on the ecosystem of antibiotic residues in the environment have become a global concern. However, little is known about the transformation mechanism of antibiotics by manganese peroxidase (MnP) from microorganisms. This work investigated the transformation characteristics, the antibacterial activity of byproducts, and the degradation mechanism of tetracycline (TC) by purified MnP from Phanerochaete chrysosporium. The results show that nitrogen-limited and high level of Mn2+ medium could obtain favorable MnP activity and inhibit the expression of lignin peroxidase by Phanerochaete chrysosporium. The purified MnP could transform 80% tetracycline in 3 h, and the threshold of reaction activator (H2O2) was about 0.045 mmol L-1. After the 3rd cyclic run, the transformation rate was almost identical at the low initial concentration of TC (77.05-88.47%), while it decreased when the initial concentration was higher (49.36-60.00%). The antimicrobial potency of the TC transformation products by MnP decreased throughout reaction time. We identified seven possible degradation products and then proposed a potential TC transformation pathway, which included demethylation, oxidation of the dimethyl amino, decarbonylation, hydroxylation, and oxidative dehydrogenation. These findings provide a novel comprehension of the role of MnP on the fate of antibiotics in nature and may develop a potential technology for tetracycline removal.
| S-EPMC8619068 | biostudies-literature
Project description:We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.
| S-EPMC1219140 | biostudies-other
Project description:Manganese peroxidase (MnP) is a major, extracellular component of the lignin-degrading system produced by the wood-rotting basidiomycetous fungus Phanerochaete chrysosporium. The transcription of MnP-encoding genes (mnps) in P. chrysosporium occurs as a secondary metabolic event, triggered by nutrient-nitrogen limitation. In addition, mnp expression occurs only under Mn2+ supplementation. Using a reporter system based on the enhanced green fluorescent protein gene (egfp), we have characterized the P. chrysosporium mnp1 promoter by examining the effects of deletion, replacement, and translocation mutations on mnp1 promoter-directed egfp expression. The 1,528-bp mnp1 promoter fragment drives egfp expression only under Mn2+-sufficient, nitrogen-limiting conditions, as required for endogenous MnP production. However, deletion of a 48-bp fragment, residing 521 bp upstream of the translation start codon in the mnp1 promoter, or replacement of this fragment with an unrelated sequence resulted in egfp expression under nitrogen limitation, both in the absence and presence of exogenous Mn2+. Translocation of the 48-bp fragment to a site 120 bp downstream of its original location resulted in Mn2+-dependent egfp expression under conditions similar to those observed with the wild-type mnp1 promoter. These results suggest that the 48-bp fragment contains at least one Mn2+-responsive cis element. Additional promoter-deletion experiments suggested that the Mn2+ element(s) is located within the 33-bp sequence at the 3' end of the 48-bp fragment. This is the first promoter sequence containing a Mn2+-responsive element(s) to be characterized in any eukaryotic organism.
| S-EPMC420142 | biostudies-literature
Project description:Manganese peroxidase (MnP) is an extracellular heme enzyme produced by the lignin-degrading white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the peroxide-dependent oxidation of Mn(II) to Mn(III). The Mn(III) is released from the enzyme in complex with oxalate, enabling the oxalate-Mn(III) complex to serve as a diffusible redox mediator capable of oxidizing lignin, especially under the mediation of unsaturated fatty acids. One heme propionate and the side chains of Glu35, Glu39 and Asp179 have been identified as Mn(II) ligands in our previous crystal structures of native MnP. In our current work, new 0.93A and 1.05A crystal structures of MnP with and without bound Mn(II), respectively, have been solved. This represents only the sixth structure of a protein of this size at 0.93A resolution. In addition, this is the first structure of a heme peroxidase from a eukaryotic organism at sub-Angstrom resolution. These new structures reveal an ordering/disordering of the C-terminal loop, which is likely required for Mn binding and release. In addition, the catalytic Arg42 residue at the active site, normally thought to function only in the peroxide activation process, also undergoes ordering/disordering that is coupled to a transient H-bond with the Mn ligand, Glu39. Finally, these high-resolution structures also reveal the exact H atoms in several parts of the structure that are relevant to the catalytic mechanism.
| S-EPMC2866031 | biostudies-literature
Project description:Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~-180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, and measurements made in a bis-tris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52, and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential are also explored as a pathway for model building.
| S-EPMC3596509 | biostudies-literature
Project description:DypB from Rhodococcus jostii RHA1 is a bacterial dye-decolorizing peroxidase (DyP) that oxidizes lignin and Mn(II). Three residues interact with the iron-bound solvent species in ferric DypB: Asn-246 and the conserved Asp-153 and Arg-244. Substitution of either Asp-153 or Asn-246 with alanine minimally affected the second order rate constant for Compound I formation (k(1) ∼ 10(5) M(-1)s(-1)) and the specificity constant (k(cat)/K(m)) for H(2)O(2). Even in the D153A/N246A double variant, these values were reduced less than 30-fold. However, these substitutions dramatically reduced the stability of Compound I (t(1/2) ∼ 0.13 s) as compared with the wild-type enzyme (540 s). By contrast, substitution of Arg-244 with leucine abolished the peroxidase activity, and heme iron of the variant showed a pH-dependent transition from high spin (pH 5) to low spin (pH 8.5). Two variants were designed to mimic the plant peroxidase active site: D153H, which was more than an order of magnitude less reactive with H(2)O(2), and N246H, which had no detectable peroxidase activity. X-ray crystallographic studies revealed that structural changes in the variants are confined to the distal heme environment. The data establish an essential role for Arg-244 in Compound I formation in DypB, possibly through charge stabilization and proton transfer. The principle roles of Asp-153 and Asn-246 appear to be in modulating the subsequent reactivity of Compound I. These results expand the range of residues known to catalyze Compound I formation in heme peroxidases.
| S-EPMC3323036 | biostudies-literature
Project description:Background and aimsPlants have evolved a number of inducible defence mechanisms against pathogen attack, including synthesis of pathogenesis-related proteins. The aim of the study was to purify and characterize antifungal protein from leaves of Acorus calamus.MethodsLeaf proteins from A. calamus were fractionated by cation exchange chromatography and gel filtration and the fraction inhibiting the hyphal extension of phytopathogens was characterized. The temperature stability and pH optima of the protein were determined and its presence was localized in the leaf tissues.Key resultsThe purified protein was identified as a class III haem peroxidase with a molecular weight of approx. 32 kDa and pI of 7.93. The temperature stability of the enzyme was observed from 5 degrees C to 60 degrees C with a temperature optimum of 36 degrees C. Maximum enzyme activity was registered at pH 5.5. The pH and temperature optima were corroborated with the antifungal activity of the enzyme. The enzyme was localized in the leaf epidermal cells and lumen tissues of xylem, characteristic of class III peroxidases. The toxic nature of the enzyme which inhibited hyphal growth was demonstrated against phytopathogens such as Macrophomina phaseolina, Fusarium moniliforme and Trichosporium vesiculosum. Microscopic observations revealed distortion in the hyphal structure with stunted growth, increased volume and extensive hyphal branching.ConclusionsThis study indicates that peroxidases may have a role to play in host defence by inhibiting the hyphal extension of invading pathogens.
| S-EPMC2803583 | biostudies-literature
Project description:Skin darkening results as a consequence of the accumulation of skin pigment melanin. To combat this, the amplitude of skin lightening agents are commercially available, most of which inhibit melanin synthesis. Decolorization of melanin is an alternative method of skin lightening. In this study, we show that lignin peroxidase (LiP), an extracellular enzyme purified from Phanerochaete chrysosporium NK-1 isolated from a forest soil can effectively degrade and decolorize melanin in vitro. Decolorization conditions including pH, temperature, incubation time, enzyme concentration, and mediator addition were investigated to optimize the reaction conditions. The results indicate that pH 3, 40 °C, 15 IU/ml, and 10 h incubation were the optimal conditions for the decolorization of the melanin. The use of the mediator, veratryl alcohol was also found effective to enhance the efficacy of the melanin decolonization, with up to 92% decolorization. The scanning electron microscopy results showed void spaces on the treated melanin granules as compared to the untreated sample, indicating the degradation of melanin. Changes in the fingerprint region of the melanin were observed. Between wavenumbers 1500-500 cm-1, for example, the presence of new peaks in the treated melanin at 1513, 1464, and 1139 cm-1 CH2, CH3 bend and C-O-C stretch represented structural changes. A new peak at 2144 cm-1 (alkynyl C?C stretch) was also detected in the decolorized melanin. The cytotoxicity study has shown that the treated melanin and LiP have low cytotoxic effects; however, the mediator of veratryl alcohol could result in high mortality which suggests that its use should be meticulously tested in formulating health and skincare products. The findings of the study suggest that LiP produced by Phanerochaete chrysosporium has the potential to be used in the medical and cosmetic industries, particularly for the development of biobased cosmetic whitening agents.
| S-EPMC7677534 | biostudies-literature
Project description:We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O(2)) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O(2) gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O(2) (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O(2) concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O(2) is at least partially mediated by the intracellular ROS.
| S-EPMC262269 | biostudies-literature
Project description:Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.
| S-EPMC2249845 | biostudies-literature