Schallmey Marcus, Jekel Peter, Tang Lixia, Majerić Elenkov Maja, Höffken Hans Wolfgang, Hauer Bernhard, Janssen Dick B
Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
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.
Enzyme Microb Technol. 2015 Mar;70:50-7. doi: 10.1016/j.enzmictec.2014.12.009. Epub 2014 Dec 24.
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.
卤代醇脱卤酶催化的氰化物介导的环氧化物开环反应可生成β-羟基腈，这在合成化学中具有很高的研究价值。迄今为止，研究最为深入的卤代醇脱卤酶是来自放射形土壤杆菌的酶，但该酶（HheC）仅表现出较低的氰解活性。对来自棒状杆菌属和分枝杆菌属的一对相关卤代醇脱卤酶进行序列比较后发现，与活性位点相互作用的苏氨酸的取代可能是导致前一种酶具有较高氰解活性的原因。在此我们报道，采用这种取代（T134A）的HheC变体在氰化物介导的环氧化物开环反应中表现出高达11倍的更高活性。该突变导致134位残基与活性位点丝氨酸132的侧链O之间氢键的消除，而丝氨酸132会向底物氧提供氢键。该突变还提高了对各种底物的脱卤酶反应速率。结构分析表明，突变酶的阴离子结合位点保持不变，这表明活性增强是由于与底物氧的相互作用改变，而非亲核试剂结合位点的变化。
