title: A single point mutation enhances hydroxynitrile synthesis by halohydrin dehalogenase.
abstract: The cyanide-mediated ring opening of epoxides catalyzed by halohydrin dehalogenases yields β-hydroxynitriles that are of high interest for synthetic chemistry. The best studied halohydrin dehalogenase to date is the enzyme from Agrobacterium radiobacter, but this enzyme (HheC) exhibits only low cyanolysis activities. Sequence comparison between a pair of related halohydrin dehalogenases from Corynebacterium and Mycobacterium suggested that substitution of a threonine that interacts with the active site might be responsible for the higher cyanolytic activity of the former enzyme. Here we report that a variant of HheC in which this substitution (T134A) is adopted displays an up to 11-fold higher activity in cyanide-mediated epoxide ring-opening. The mutation causes removal of the hydrogen bond between residue 134 and the side chain O of the active site serine 132, which donates a hydrogen bond to the substrate oxygen. The mutation also increases dehalogenase rates with various substrates. Structural analysis revealed that the anion-binding site of the mutant enzyme remained unaltered, showing that the enhanced activity is due to altered interactions with the substrate oxygen rather than changes in the nucleophile binding site.
authors: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors: Marcus
authors: M
authors: Schallmey
authors: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors: Peter
authors: P
authors: Jekel
authors: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China. Electronic address: lixiatang@uestc.edu.cn.
authors: Lixia
authors: L
authors: Tang
authors: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia. Electronic address: majeric@irb.hr.
authors: Maja
authors: M
authors: Majerić Elenkov
authors: BASF SE, Biological & Effect Systems Research, Ludwigshafen, Germany. Electronic address: wolfgang.hoeffken@basf.com.
authors: Hans Wolfgang
authors: HW
authors: Höffken
authors: BASF SE, Biological & Effect Systems Research, Ludwigshafen, Germany. Electronic address: bernhard.hauer@itb.uni-stuttgart.de.
authors: Bernhard
authors: B
authors: Hauer
authors: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Electronic address: d.b.janssen@rug.nl.
authors: Dick B
authors: DB
authors: Janssen
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology; D020134:Catalytic Domain / Q000235:genetics; D060326:Chemistry Techniques, Synthetic; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*; D010069:Oxadiazoles; D017354:Point Mutation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009570:Nitriles; D010069:Oxadiazoles; C050143:MO 113; D006867:Hydrolases; C430848:halohydrin dehalogenase
keywords: Cyanide; Epoxides; Halohydrin dehalogenase; Hydroxynitrile; Ligand binding; Protein engineering