Title
Examining the role of glutamic acid 183 in chloroperoxidase catalysis
Author
TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Yi, X.
Conesa, A.
Punt, P.J.
Hager, L.P.
Publication year
2003
Abstract
Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed in Aspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced ∼2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site.
Subject
Biotechnology
Catalysis
Genetic engineering
Organic acids
X ray crystallography
Active sites
Enzymes
Chloride peroxidase
Clutamic acid
Histidine
Hydrogen peroxide
Natural product
Epoxidase
Oxidoreductase
Amino acid substitution
Animal cell
Asymmetric catalysis
Chlorination
Enzyme active site
Enzyme activity
Epoxidation
Genetic analysis
Hydrophobicity
Nonhuman
Plasmid
Site directed mutagenesis
X ray crystallography
Chemistry
Circular dichroism
Enzymology
Immunoblotting
Ion exchange chromatography
Isoelectric focusing
Metabolism
PH
polyacrylamide gel electrophoresis
Animalia
Aspergillus
Aspergillus niger
Catalase
Catalysis
Chloride Peroxidase
Chlorine
Chromatography, Ion Exchange
Circular Dichroism
Crystallography, X-Ray
Electrophoresis, Polyacrylamide Gel
Fungi
Glutamic Acid
Histidine
Hydrogen-Ion Concentration
Immunoblotting
Isoelectric Focusing
Mutation
Oxidoreductases
Plasmids
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http://resolver.tudelft.nl/uuid:814c819f-e25c-4df7-879f-fa3ef3ceb148
DOI
https://doi.org/10.1074/jbc.m210906200
TNO identifier
237045
ISSN
0021-9258
Source
Journal of Biological Chemistry, 278 (278), 13855-13859
Document type
article