Computational Library Design for Increasing Haloalkane Dehalogenase Stability

Robert J. Floor, Hein J. Wijma, Dana I. Colpa, Aline Ramos-Silva, Peter A. Jekel, Wiktor Szymanski, Ben L. Feringa, Siewert J. Marrink, Dick B. Janssen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

41 Citations (Scopus)

Abstract

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 degrees C increase in apparent melting temperature (T-m,T-app, 72.5 degrees C) and an over 200-fold longer half-life at 60 degrees C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

Original languageEnglish
Pages (from-to)1659-1671
Number of pages13
JournalChemBioChem
Volume15
Issue number11
DOIs
Publication statusPublished - 21-Jul-2014

Keywords

  • computational design
  • co-solvents
  • dehalogenases
  • directed evolution
  • thermostability
  • virtual screening
  • SPHINGOMONAS-PAUCIMOBILIS UT26
  • MOLECULAR-DYNAMICS SIMULATIONS
  • SITE-DIRECTED MUTAGENESIS
  • PROTEIN STABILITY
  • ENZYME STABILITY
  • SOLVENT-RESISTANT
  • CRYSTAL-STRUCTURE
  • GAMMA-HEXACHLOROCYCLOHEXANE
  • ENHANCED THERMOSTABILITY
  • INTRINSIC DISORDER

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