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Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid.
- Source :
-
The Biochemical journal [Biochem J] 2021 May 14; Vol. 478 (9), pp. 1795-1808. - Publication Year :
- 2021
-
Abstract
- To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.<br /> (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Subjects :
- Amino Acid Substitution
Biocatalysis
Catalytic Domain
Cloning, Molecular
Dihydroxyphenylalanine genetics
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Genetic Vectors chemistry
Genetic Vectors metabolism
Heme metabolism
Histidine genetics
Humans
Hydrogen Bonding
Hydrogen Peroxide chemistry
Hydrogen Peroxide metabolism
Iron metabolism
Kinetics
Models, Molecular
Myoglobin chemistry
Myoglobin genetics
Oxidation-Reduction
Peroxidases chemistry
Peroxidases metabolism
Protein Binding
Protein Conformation
Protein Engineering methods
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Dihydroxyphenylalanine metabolism
Heme chemistry
Histidine metabolism
Iron chemistry
Myoglobin metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1470-8728
- Volume :
- 478
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 33821889
- Full Text :
- https://doi.org/10.1042/BCJ20210091