Structure, biochemical characterization and analysis of the pleomorphism of carboxylesterase Cest-2923 from Lactobacillus plantarum WCFS1
Structure, biochemical characterization and analysis of the pleomorphism of carboxylesterase Cest-2923 from Lactobacillus plantarum WCFS1
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DOI：
10.1111/febs.12569
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发表时间：
2013-12-01
期刊：
影响因子：
5.4
通讯作者：
Mancheno, Jose M.
中科院分区：
文献类型：
--
作者：
Benavente, Rocio;Esteban-Torres, Maria;Mancheno, Jose M.
根据采用这种三维结构的序列的多样性，水解酶折叠是蛋白质领域中最通用的结构之一。在本研究中，我们澄清的羧酸酯酶Cest-2923从乳酸菌Lactobacillusplantarum WCFS1精制到2.1埃分辨率的晶体结构，确定其主要的生化特性，并进行了分析，其在溶液中的缔合行为。我们发现，一个典型的/水解酶折叠，Cest-2923的晶体结构的基本框架的多功能性，也延伸到其在溶液中的低聚行为。因此，我们发现Cest-2923在涉及单体、规范二聚体和四聚体的溶液中表现出pH依赖性的多晶行为。虽然，在中性pH下，该系统主要转移到二聚体物种，在酸性条件下，四聚体物种占主导地位。尽管这些四聚体是由典型二聚体的缔合产生的，如在来自对脂肪酶敏感的脂肪酶家族的许多其他羧酸酯酶中常见的，但它们可以被定义为"非典型的"，因为它们代表不同的缔合模式。我们在Cest-2923的最近亲属中鉴定了这种相同类型的四聚体，其结构特征为：来自乳酸乳球菌的糖水解酶YeeB。所观察到的关联行为与来自结构基因组学联合体的Cest-2923的不同晶体学结果一致。最后，硫酸盐或乙酸盐分子的存在（取决于所分析的晶体形式）在亲核体Ser 116附近的相互作用使我们能够鉴定与推定的氧阴离子空穴的相互作用并推断Cest-2923晶体内水解活性的存在。结构化数字摘要Cest-2923和Cest-2923通过X射线晶体学结合（1，2）Cest-2923和Cest-2923在溶液中通过共沉淀结合（1，2）数据库原子坐标和结构因子已经以登录号4BZW保藏在蛋白质数据库中，Cest-2923来自未用底物浸泡的天然晶体（P6（3）22空间群）; Cest-2923为4C01，来自用乙酸苯酯（C2空间群）浸泡的晶体; Cest-2923为4BZZ，来自用乙酸异丙烯酯（P622空间群）浸泡的晶体。
The hydrolase fold is one of the most versatile structures in the protein realm according to the diversity of sequences adopting such a three-dimensional architecture. In the present study, we clarified the crystal structure of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillusplantarum WCFS1 refined to 2.1 angstrom resolution, determined its main biochemical characteristics and also carried out an analysis of its associative behaviour in solution. We found that the versatility of a canonical /hydrolase fold, the basic framework of the crystal structure of Cest-2923, also extends to its oligomeric behaviour in solution. Thus, we discovered that Cest-2923 exhibits a pH-dependent pleomorphic behaviour in solution involving monomers, canonical dimers and tetramers. Although, at neutral pH, the system is mainly shifted to dimeric species, under acidic conditions, tetrameric species predominate. Despite these tetramers resulting from the association of canonical dimers, as is commonly found in many other carboxylesterases from the hormone-sensitive lipase family, they can be defined as noncanonical' because they represent a different association mode. We identified this same type of tetramer in the closest relative of Cest-2923 that has been structurally characterized: the sugar hydrolase YeeB from Lactococcuslactis. The observed associative behaviour is consistent with the different crystallographic results for Cest-2923 from structural genomics consortia. Finally, the presence of sulfate or acetate molecules (depending on the crystal form analysed) in the close vicinity of the nucleophile Ser116 allows us to identify interactions with the putative oxyanion hole and deduce the existence of hydrolytic activity within Cest-2923 crystals.Structured digital abstract Cest-2923 and Cest-2923 bind by x-ray crystallography (1, 2)Cest-2923 and Cest-2923 bind by cosedimentation in solution (1, 2)Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 4BZW for Cest-2923 from native crystals not soaked with substrates (P6(3)22 space group); 4C01 for Cest-2923 from crystals soaked with phenyl acetate (C2 space group); 4BZZ for Cest-2923 from crystals soaked with isopropenyl acetate (P622 space group).