PDF(Pubmed)
Abstract：
Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5\'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.
摘要：
具有扩展的底物特异性的酶是设计用于目标反应的生物催化剂的良好起点。然而,扩展的底物特异性的结构基础仍然难以捉摸，尤其是在吡哆醛-5'-磷酸依赖性转氨酶超家族中，其特征在于活性位点和功能性二聚体的保守组织。这里,我们分析了来自萨氏芽孢球菌的非标准D-氨基酸转氨酶的结构-功能关系，对D-氨基酸和伯(R)-胺有活性。对该酶的详细研究包括对其底物范围的动力学分析以及对全酶及其与苯肼的复合物的结构分析-苯肼是（R）-1-苯乙胺的可逆抑制剂和类似物-（R）-选择性胺转氨酶的基准底物。我们建议，从B.saxobsidens转氨酶的活性位点的特征，例如R34和R96残基的灵活性，在活性位点入口处的β-转角中缺乏庞大的残基，和短O形口袋环，促进具有和不具有α-羧酸酯基团的底物的结合。所提出的扩展的底物特异性的结构决定因素可用于设计用于酮化合物的立体选择性胺化的转氨酶。
