title: A robust cosolvent-compatible halohydrin dehalogenase by computational library design.
abstract: To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Hesam
authors.initials: H
authors.lastname: Arabnejad
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Marco
authors.initials: M
authors.lastname: Dal Lago
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Jekel
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Floor
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Andy-Mark W H
authors.initials: AWH
authors.lastname: Thunnissen
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Terwisscha van Scheltinga
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Hein J
authors.initials: HJ
authors.lastname: Wijma
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D019943:Amino Acid Substitution; D003198:Computer Simulation; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D020125:Mutation, Missense; D017510:Protein Folding
chemical_list: D006867:Hydrolases; C430848:halohydrin dehalogenase
keywords: biocatalysis; cosolvent stability; haloalcohol dehalogenase; protein engineering; thermostability