Selecting of a cytochrome P450cam SeSaM library with 3-Chloroindole and Endosulfan – Identification of mutants that Dehalogenate 3-Chloroindole

Publication date: Available online 18 September 2017
Source:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Author(s): Shaima Kammoonah, Brinda Prasad, Priyadarshini Balaraman, Hemanshu Mundhada, Ulrich Schwaneberg, Erika Plettner
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.List of Chemical Compounds.1(R) (+)-camphor (PubChem CID 159055); 3-chloroindole (PubChem CID 177790); endosulfan (PubChem CID 3224); indigo (PubChem CID 5318432); isatin (PubChem CID 7054); m-chloroperbenzoic acid, m-CPBA (PubChem ID 70297); tricarbonylchloro(glycinato)ruthenium, CORM-3 (PubChem ID 91886169).



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