Structure of the active site of lignin peroxidase isozyme H2: native enzyme, compound III and reduced form

Biochemistry ◽  
1992 ◽  
Vol 31 (20) ◽  
pp. 4892-4900 ◽  
Author(s):  
R. Sinclair ◽  
I. Yamazaki ◽  
J. Bumpus ◽  
B. Brock ◽  
C. S. Chang ◽  
...  
Keyword(s):  
Lignin Peroxidase ◽  
Active Site ◽  
Native Enzyme ◽  
Reduced Form ◽  
Peroxidase Isozyme

Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium

1989 ◽  
Vol 9 (6) ◽  
pp. 2743-2747
Author(s):  
H Schalch ◽  
J Gaskell ◽  
T L Smith ◽  
D Cullen
Keyword(s):  
Molecular Cloning ◽  
Phanerochaete Chrysosporium ◽  
Lignin Peroxidase ◽  
Nucleotide Sequences ◽  
Peroxidase Isozyme ◽  
Genomic Clones

The genomic clones encoding lignin peroxidase isozyme H8 and two closely related genes were isolated from Phanerochaete chrysosporium BKM-1767, and their nucleotide sequences were determined. The positions and approximate lengths of introns were found to be highly conserved in all three clones. Analysis of homokaryotic derivatives indicated that the three clones are not alleles of the same gene(s).

Solution Structure of a Natural CPPC Active Site Variant, the Reduced Form of Thioredoxinh1 from Poplar†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (6) ◽  
pp. 2001-2008 ◽  
Author(s):  
Nicolas Coudevylle ◽  
Aurélien Thureau ◽  
Christine Hemmerlin ◽  
Eric Gelhaye ◽  
Jean-Pierre Jacquot ◽  
...  
Keyword(s):  
Active Site ◽  
Solution Structure ◽  
Reduced Form

scholarly journals Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding

1996 ◽  
Vol 314 (3) ◽  
pp. 985-991 ◽  
Author(s):  
Subrata ADAK ◽  
Abhijit MAZUMDER ◽  
Ranajit K. BANERJEE
Keyword(s):  
Active Site ◽  
Native Enzyme ◽  
Tyrosine Residue ◽  
Tyrosine Residues ◽  
First Order ◽  
First Order Kinetics ◽  
Aromatic Donor ◽  
Pseudo First Order ◽  
Modified Enzyme ◽  
Compound Ii

The plausible role of arginine and tyrosine residues at the active site of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate constants of 24 M-1·min-1, 0.8 M-1·min-1 and 0.54 M-1·min-1 respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0 M-1·min-1. The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement of arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (∆G´) for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete inactivation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35 mM compared with 10 mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichroism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.

Human Prostatic Acid Phosphatase: Properties of the Native Enzyme, and the Enzyme-Antibody Complex

1983 ◽  
Vol 20 (6) ◽  
pp. 374-380 ◽  
Author(s):  
Renze Bais ◽  
Anne Huxtable ◽  
John B Edwards
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostatic Acid Phosphatase ◽  
Native Enzyme ◽  
Terminal Amino Acid ◽  
Large Molecular Weight ◽  
Antibody Complex ◽  
Terminal Amino

Acid phosphatase purified from human prostatic tissue was shown to be homogeneous by polyacrylamide gel electrophoresis and N-terminal amino acid analysis. However, isoelectric focusing revealed a large number of isoenzymes which were reduced to four by digestion with neuraminidase. It is suggested that the patterns observed are due to differences in bound carbohydrate attached to the same protein backbone. Antiserum to the purified enzyme was produced in rabbits and reacted with the enzyme to form an enzymatically active complex of large molecular weight. This complex is more stable at high temperatures than the native enzyme. Kinetic analysis of both the enzyme and the enzyme-antibody complex demonstrated that the binding of the antibody caused no significant change to the active site of the enzyme.

scholarly journals Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

2009 ◽  
Vol 284 (24) ◽  
pp. 16264-16276 ◽  
Author(s):  
Miguel Saggu ◽  
Ingo Zebger ◽  
Marcus Ludwig ◽  
Oliver Lenz ◽  
Bärbel Friedrich ◽  
...  
Keyword(s):  
Active Site ◽  
Ralstonia Eutropha ◽  
Large Subunit ◽  
Magnetic Coupling ◽  
Spectroscopic Characterization ◽  
Spectroscopic Properties ◽  
Small Subunit ◽  
Reduced Form ◽  
Strictly Anaerobic ◽  
Redox States

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

scholarly journals Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

2021 ◽  
Author(s):  
Le Thanh Mai Pham ◽  
Kai Deng ◽  
Trent R Northen ◽  
Steven W Singer ◽  
Paul D Adams ◽  
...  
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Lignin Peroxidase ◽  
Bond Cleavage ◽  
Product Distribution ◽  
Bond Breaking ◽  
Hydroxyl Groups ◽  
Elastic Band ◽  
Theoretical Understanding ◽  
Peroxidase Isozyme ◽  
Lignin Peroxidases

Abstract Background:Lignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4’ ether bonds and C–C bonds in lignin, both of which are essential for depolymerizing lignin into fragments amendable to biological or chemical upgrading to valuable products. Studies of the specificity of lignin peroxidases to catalyze these various reactions and the role reaction conditions such as pH play have been limited by the lack of assays that allow quantification of specific bond-breaking events. The subsequent theoretical understanding of the underlying mechanisms by which pH modulates the activity of lignin peroxidases remains nascent. Here, we report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-O-4’ ether bonds and of C–C bonds by a lignin peroxidase isozyme H8 from Phanerochaete chrysosporium and an acid stabilized variant of the same enzyme.Results: Using a nanostructure initiator mass spectrometry assay that provides quantification of bond breaking in a phenolic model lignin dimer we found that catalysis of degradation of the dimer to products by an acid-stabilized variant of lignin peroxidase isozyme H8 increased from 38.4 % at pH 5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution resulted from 65.5% b-O-4’ ether bond cleavage, 27.0% Ca-C1 carbon bond cleavage and 3.6% Ca-oxidation as by-product. Using ab initio molecular dynamic simulations and climbing-image Nudge Elastic Band based transition state searches, we suggest the effect of lower pH is via protonation of aliphatic hydroxyl groups under which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, particularly β-O-4’ bonds. Conclusion: These coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved lignin depolymerization should include targeting stability at low pH.

Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase

2020 ◽  
Vol 76 (10) ◽  
pp. 946-953
Author(s):  
Takeshi Hiromoto ◽  
Koji Nishikawa ◽  
Seiya Inoue ◽  
Hiroaki Matsuura ◽  
Yu Hirano ◽  
...  
Keyword(s):  
Neutron Diffraction ◽  
Diffraction Data ◽  
Active Site ◽  
National Laboratory ◽  
Reduced Form ◽  
Desulfovibrio Vulgaris ◽  
X Rays ◽  
Oak Ridge ◽  
Fe Complex ◽  
Cryogenic Conditions

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni–Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni–Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni–Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

Microbial hydrogen splitting in the presence of oxygen

2013 ◽  
Vol 41 (5) ◽  
pp. 1317-1324 ◽  
Author(s):  
Matthias Stein ◽  
Sandeep Kaur-Ghumaan
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Spectroscopic Studies ◽  
Small Subunit ◽  
Reduced Form ◽  
Cysteine Residues ◽  
Binding Motif ◽  
Oxygen Tolerance ◽  
Symmetry Approach ◽  
Long Time

The origin of the tolerance of a subclass of [NiFe]-hydrogenases to the presence of oxygen was unclear for a long time. Recent spectroscopic studies showed a conserved active site between oxygen-sensitive and oxygen-tolerant hydrogenases, and modifications in the vicinity of the active site in the large subunit could be excluded as the origin of catalytic activity even in the presence of molecular oxygen. A combination of bioinformatics and protein structural modelling revealed an unusual co-ordination motif in the vicinity of the proximal Fe–S cluster in the small subunit. Mutational experiments confirmed the relevance of two additional cysteine residues for the oxygen-tolerance. This new binding motif can be used to classify sequences from [NiFe]-hydrogenases according to their potential oxygen-tolerance. The X-ray structural analysis of the reduced form of the enzyme displayed a new type of [4Fe–3S] cluster co-ordinated by six surrounding cysteine residues in a distorted cubanoid geometry. The unusual electronic structure of the proximal Fe–S cluster can be analysed using the broken-symmetry approach and gave results in agreement with experimental Mößbauer studies. An electronic effect of the proximal Fe–S cluster on the remote active site can be detected and quantified. In the oxygen-tolerant hydrogenases, the hydride occupies an asymmetric binding position in the Ni-C state. This may rationalize the more facile activation and catalytic turnover in this subclass of enzymes.

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