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

Resource type
Date created
2017-09
Authors/Contributors
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.
Document
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
Publication title
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Document title
Selecting of a Cytochrome P450cam SeSaM Library with 3-Chloroindole and Endosulfan - Identification of Mutants that Dehalogenate 3-Chloroindole
Date
2018
First page
68
Last page
79
Publisher DOI
10.1016/j.bbapap.2017.09.006
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Copyright is held by the author(s).
Scholarly level
Peer reviewed?
Yes
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