Lignin peroxidase structure and function

2001 ◽  
Vol 29 (2) ◽  
pp. 111-116 ◽  
Author(s):  
K. Piontek ◽  
A. T. Smith ◽  
W. Blodig
Keyword(s):  
Lignin Peroxidase ◽  
Structural Changes ◽  
Plant Cell Wall ◽  
Peptide Mapping ◽  
Spin Trapping ◽  
Site Directed Mutagenesis ◽  
Artificial Substrates ◽  
Protein Chemistry ◽  
Redox Potentials ◽  
Radical Intermediate

Lignin peroxidase (LiP) plays a central role in the biodegradation of the plant cell wall constituent lignin. LiP is able to oxidize aromatic compounds with redox potentials higher than 1.4 V (NHE) by single electron abstraction, but the exact redox mechanism is still poorly understood. The finding in our laboratory that the Cβ-atom of Trp171 carries a unique modification led us to initiate experiments to investigate the role of this residue. These experiments, employing crystallography, site-directed mutagenesis, protein chemistry, spin-trapping and spectroscopy, yielded the following results: (i) Trp171 is stereospecifically hydroxylated at its Cβ-atom as the result of an auto-catalytic process, which occurs under turnover conditions in the presence of hydrogen peroxide, (ii) Evidence for the formation of a Trp171 radical intermediate has been obtained using spin-trapping, in combination with peptide mapping and protein crystallography. (iii) Trp171 is very likely to be involved in electron transfer from natural substrates to the haem cofactor via LRET. (iv) Mutagenetic substitution of Trp171 abolishes completely the oxidation activity for veratryl alcohol, but not for artificial substrates. (v) Structural changes in response to the mutation are marginal. Therefore the lack of activity is due to the absence of the redox active indole side chain.

scholarly journals Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 Å resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate

1998 ◽  
Vol 335 (2) ◽  
pp. 409-416 ◽  
Author(s):  
Lloyd F. MACKENZIE ◽  
Gerlind SULZENBACHER ◽  
Christina DIVNE ◽  
T. Alwyn JONES ◽  
Helle F. WÖLDIKE ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Critical Role ◽  
Soft Rot ◽  
Site Directed Mutagenesis ◽  
Cellobiohydrolase I ◽  
Native Form ◽  
Humicola Insolens ◽  
Endoglucanase I ◽  
Enzyme Intermediate

Cellulose is the major polysaccharide component of the plant cell wall and the most abundant naturally produced macromolecule on Earth. The enzymic degradation of cellulose, by cellulases, is therefore of great environmental and commercial significance. Cellulases are found in 12 of the glycoside hydrolase families classified according to their amino acid sequence similarities. Endoglucanase I (Cel7B), from the soft-rot fungus Humicola insolens, is a family 7 enzyme. The structure of the native form of Cel7B from H. insolens at 2.2 Å resolution has been solved by molecular replacement using the known Trichoderma reesei cellobiohydrolase I [Divne, Ståhlberg, Reinikainen, Ruohonen, Pettersson, Knowles, Teeri and Jones (1994) Science265, 524–528] structure as the search model. Cel7B catalyses hydrolysis of the β-1,4 glycosidic linkages in cellulose with net retention of anomeric configuration. The catalytic nucleophile at the active site of Cel7B has been identified as Glu-197 by trapping of a 2-deoxy-2-fluorocellotriosyl enzyme intermediate and identification of the labelled peptide in peptic digests by tandem MS. Site-directed mutagenesis of both Glu-197 and the prospective catalytic acid, Glu-202, results in inactive enzyme, confirming the critical role of these groups for catalysis.

Evidence from Spin-Trapping for a Transient Radical on Tryptophan Residue 171 of Lignin Peroxidase

1999 ◽  
Vol 370 (1) ◽  
pp. 86-92 ◽  
Author(s):  
Wolfgang Blodig ◽  
Andrew T. Smith ◽  
Kaspar Winterhalter ◽  
Klaus Piontek
Keyword(s):  
Lignin Peroxidase ◽  
Spin Trapping ◽  
Tryptophan Residue

scholarly journals Structural and biochemical basis of the formation of isoaspartate in the complementarity-determining region of antibody 64M-5 Fab

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hideshi Yokoyama ◽  
Ryuta Mizutani ◽  
Shuji Noguchi ◽  
Naoki Hayashida
Keyword(s):  
Normal Form ◽  
Electrostatic Interactions ◽  
Structural Changes ◽  
Peptide Mapping ◽  
Exchange Chromatography ◽  
Side Chain ◽  
Type I ◽  
Biochemical Basis ◽  
Structural Conversion ◽  
Complementarity Determining Region

AbstractThe formation of the isoaspartate (isoAsp) is one of spontaneous degradation processes of proteins, affecting their stability and activity. Here, we report for the first time the crystal structures of an antibody Fab that contains isoAsp in the complementarity-determining region (CDR), along with biochemical studies to detect isoAsp. By comparing the elution profiles of cation-exchange chromatography, it was clarified that the antibody 64M-5 Fab is converted from the normal form to isoAsp form spontaneously and time-dependently under physiological conditions. The isoAsp residue was identified with tryptic peptide mapping, N-terminal sequencing, and the protein isoaspartyl methyltransferase assay. Based on the fluorescence quenching method, the isoAsp form of 64M-5 Fab shows a one order of magnitude lower binding constant for its dinucleotide ligand dT(6–4)T than the normal form. According to the structure of the isoAsp form, the conformation of CDR L1 is changed from the normal form to isoAsp form; the loss of hydrogen bonds involving the Asn28L side-chain, and structural conversion of the β-turn from type I to type II’. The formation of isoAsp leads to a large displacement of the side chain of His27dL, and decreased electrostatic interactions with the phosphate group of dT(6–4)T. Such structural changes should be responsible for the lower affinity of the isoAsp form for dT(6–4)T than the normal form. These findings may provide insight into neurodegenerative diseases (NDDs) and related diseases caused by misfolded proteins.

Two Substrate Interaction Sites in Lignin Peroxidase Revealed by Site-Directed Mutagenesis†

Biochemistry ◽  
1998 ◽  
Vol 37 (43) ◽  
pp. 15097-15105 ◽  
Author(s):  
Wendy A. Doyle ◽  
Wolfgang Blodig ◽  
Nigel C. Veitch ◽  
Klaus Piontek ◽  
Andrew T. Smith
Keyword(s):  
Lignin Peroxidase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Interaction Sites ◽  
Substrate Interaction

scholarly journals Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90

1993 ◽  
Vol 296 (3) ◽  
pp. 577-583 ◽  
Author(s):  
S Basu ◽  
A Basu ◽  
M J Modak
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Structural Integrity ◽  
Peptide Mapping ◽  
Site Directed Mutagenesis ◽  
Heat Inactivation ◽  
Composition Analysis ◽  
Kinetic Measurements ◽  
Leukaemia Virus ◽  
Murine Leukaemia Virus

Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.

scholarly journals Multiple interactions of the ‘transducer’ govern its function in calpain activation by Ca2+

2005 ◽  
Vol 388 (3) ◽  
pp. 741-744 ◽  
Author(s):  
Zoltán BOZÓKY ◽  
Anita ALEXA ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Structural Changes ◽  
Catalytic Domain ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Structural Element ◽  
X Ray ◽  
Calpain Activation ◽  
Ef Hand ◽  
Domain Iii ◽  
Multiple Interactions

Typical calpains in mammals become activated on binding of 8–12 Ca2+ ions per enzyme molecule, giving an example of integrated, manifold regulation by calcium. Besides two identified Ca2+ sites in catalytic domain II and several EF-hand motifs in domains IV and VI, an acidic loop in the centrally positioned domain III seems to harbour Ca2+. The mediator of distant Ca2+-induced structural transitions is an elongated structural element, the ‘transducer’. By site-directed mutagenesis along the transducer, we have generated various forms of rat m-calpain in which critical intramolecular interactions, as judged from the X-ray structure, would be abolished or modified. The kinetic parameters of these mutant enzymes support a model featuring shrinkage of transducer as a contributor to structural changes involved in calpain activation.

Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine

1985 ◽  
Vol 133 (3) ◽  
pp. 1086-1091 ◽  
Author(s):  
Atsuko Noda ◽  
Hiroshi Noda ◽  
Kohji Ohno ◽  
Toshiaki Sendo ◽  
Ayako Misaka ◽  
...  
Keyword(s):  
Free Radical ◽  
Spin Trapping ◽  
Radical Intermediate ◽  
Microsomal Metabolism ◽  
Free Radical Intermediate
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