Journal article
Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan – Identification of mutants that dehalogenate 3-chloroindole
Simon Fraser University1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Bacterial Cell Factory Optimization, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
RWTH Aachen University5
Cytochrome P450cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin.
The wild-type enzyme did not accept 3-chloroindole as a substrate. Mutant (E156G/V247F/V253G/F256S) had the highest maximal velocity in the conversion of 3-chloroindole to isatin, whereas mutants (T56A/N116H/D297N) and (G60S/Y75H) had highest kcat/KM values. Six of the mutants had more than one mutation, and within this set, mutation of residues 297 and 179 was observed twice.
Docking simulations were performed on models of the mutant enzymes; the wild-type did not accommodate 3-chloroindole in the active site, whereas all the mutants did. We propose two potential reaction pathways for dechlorination of 3-chloroindole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
Language: | English |
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Year: | 2018 |
Pages: | 68-79 |
ISSN: | 18781454 and 15709639 |
Types: | Journal article |
DOI: | 10.1016/j.bbapap.2017.09.006 |
Amino Acid Motifs Amino Acid Substitution Bacterial Proteins Binding Sites Biodegradation, Environmental Camphor 5-Monooxygenase Cloning, Molecular Endosulfan Escherichia coli Gene Expression Gene Library Halogenation Indoles Isatin Kinetics Molecular Docking Simulation Mutation Oxidation-Reduction Protein Binding Protein Interaction Domains and Motifs Protein Structure, Secondary Pseudomonas putida Recombinant Proteins Substrate Specificity