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首页> 外文期刊>Applied Microbiology >A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (S)-Enantiopreference, and High Thermostability
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A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (S)-Enantiopreference, and High Thermostability

机译：来自Saccharomonospora viridis菌株DSM 43017的卤代烷烃脱卤素酶，一种堆肥细菌，具有异常催化残基，独特（S） - 尼尾再次引用和高热稳定性

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Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB, remain catalytically active while employing a single halide-stabilizing residue. Here, we describe a novel haloalkane dehalogenase, DsvA, from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017, possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, DsvA contains the phenylalanine F165, which cannot stabilize the halogen anion released during the enzymatic reaction by a hydrogen bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same α-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F), and DsvA03 (W162F and F165W). Interestingly, DsvA exhibits a preference for the ( S )- over the ( R )-enantiomers of β-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing an unconventional composition of catalytic residues represent a valuable source of novel biocatalysts.IMPORTANCE The present study describes a novel haloalkane dehalogenase, DsvA, originating from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and an ( S )-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. Sequence analysis revealed that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single point mutants and one double point mutant and identified the noncanonical halide-stabilizing residue. Our study underlines the importance of searching for noncanonical catalytic residues in protein sequences.
机译：卤代烷烃脱氢酶可以在宽范围的卤代脂族化合物中切割碳 - 卤素键。然而，催化活性需要由亲核试剂，催化基碱，催化酸和两个卤化物稳定残留物组成的高度保守的催化五边构。只有少数家庭成员，例如DSAA，DMXA或DMRB，在采用单一卤化物稳定残余物的同时保持催化活性。在这里，我们描述了一种新的卤代烷烃脱氢酶，来自温和的嗜热细菌，Saccharogonospora viridis菌株DSM 43017具有一种规范卤化物稳定色氨酸（W125）。在第二卤化物稳定残余物的位置，DSVA含有苯丙氨酸F165，其不能通过氢键稳定在酶促反应期间释放的卤素阴离子。基于序列和结构对准，我们鉴定了位于与F165相同的α-螺旋上的推定的第二卤化物稳定色氨酸（W162），但在活性位点的相对侧。通过三个变体，DSVA01（F165W），DSVA02（W162F）和DSVA03（W162F和F165W）的构建和生化表征来研究这种残留物在DSVA催化中的潜在累及。有趣的是，DSVA表现出对（R） - β-溴烷烃的（R） - 苯甲烷的聚体的偏好，其在任何表征卤代烷烃脱氢酶之前尚未报道。此外，DSVA在升高的温度下显示出显着的操作稳定性。本研究表明，具有催化残基的非传统组成的蛋白质序列代表了新型生物催化剂的重要来源。本研究表明了一种新的卤代烷烃脱氢酶，DSVA，源自轻度嗜热细菌，Saccharomonospora viridis菌株DSM 43017。我们报告其高温性，高温下显着的操作稳定性，和（S） - 诱发，这使得这酶成为实际应用的有吸引力的生物催化剂。序列分析显示DSVA在其活性位点中具有不寻常的卤化卤稳定色氨酸残基组成。我们构建和生物化学表征了两个单点突变体和一个双点突变体，并确定了非甘露出的卤化物稳定残余物。我们的研究强调了在蛋白质序列中寻找非碳催化残基的重要性。

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