Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan – Identification of mutants that dehalogenate 3-chloroindole

Shaima Kammoonah, Brinda Prasad, Priyadarshini Balaraman, Hemanshu Mundhada, Ulrich Schwaneberg, Erika Plettner*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

233 Downloads (Pure)

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. 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.

Original languageEnglish
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1866
Issue number1
Pages (from-to)68-79
ISSN1570-9639
DOIs
Publication statusPublished - 1 Jan 2018

Keywords

  • 3-chloroindole
  • Cytochrome P450
  • Dehalogenation
  • Enzyme kinetics
  • Oxidation
  • Random mutagenesis

Fingerprint

Dive into the research topics of 'Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan – Identification of mutants that dehalogenate 3-chloroindole'. Together they form a unique fingerprint.

Cite this