Selecting of a Cytochrome P450cam SeSaM Library with 3-Chloroindole and Endosulfan - Identification of Mutants that Dehalogenate 3-Chloroindole

Peer reviewed: 
Yes, item is peer reviewed.
Scholarly level: 
Faculty/Staff
Final version published as: 

Kammoonah, S., Prasad, B., Balaraman, P., Mundhada, H., Schwaneberg, U., and Plettner, E. (2018).Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan--Identification of mutants thatdehalogenate 3-chloroindole. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1866(1): 68-79. https://doi.org/10.1016/j.bbapap.2017.09.006

Date created: 
2017-09
Keywords: 
Cytochrome P450
Random mutagenesis
Enzyme kinetics
3-chloroindole
Oxidation
Dehalogenation
Abstract: 

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.

Description: 

The full text of this paper will be availabe in February 2019 in accordance with the embargo periods of the journal Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics.

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Language: 
English
Document type: 
Article
Rights: 
Rights remain with the authors.
Sponsor(s): 
Natural Sciences and Engineering Research Council of Canada (NSERC)
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