YesT:: A new rhamnogalacturonan acetyl esterase from Bacillus subtilis
YesT:: A new rhamnogalacturonan acetyl esterase from Bacillus subtilis
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DOI：
10.1002/prot.21705
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发表时间：
2008-04-01
影响因子：
2.9
通讯作者：
Sanchez-Ferrer, Alvaro
中科院分区：
文献类型：
--
作者：
Martinez-Martinez, Irene;Navarro-Fernandez, Jose;Sanchez-Ferrer, Alvaro
YesT是枯草芽孢杆菌ATCC 6633的一种推定蛋白，已被暂时分类为CE-12家族的鼠李糖半乳糖醛酸聚糖乙酰酯酶（RGAE），其被克隆、在大肠杆菌Rosetta（DE 3）中表达并纯化。该酶是单体，分子量为37 kDa，并呈现出类似于棘孢曲霉RGAE的嗜热特性，尽管YesT更嗜碱性。抑制剂的研究证实了除了催化三联体中的Asp外，His和亲核Ser对酯酶活性的重要性。这种酶也表现出广泛的底物特异性，并且对7-氨基头孢烷酸、头孢菌素C、乙酸对硝基苯酯、乙酸β-萘酯、葡萄糖五乙酸酯和乙酰化木聚糖具有活性。此外，YesT与木聚糖酶A一起实现了对乙酰化木聚糖的协同效应。作为SGNH家族的一员，它不采用常见的α/β水解酶折叠。一级序列分析和多重序列比对显示缺少两个β-链反平行片层，这导致结构的明显变化以及RGAE结构中存在的三个3（10）-螺旋之一的消失。本文对疥疮链霉菌RGAE、YesT和酯酶A、血小板活化因子乙酰水解酶Ib亚型α亚基[PAF-AH（Ib）α（1）]、PAF-AH（Ib）α（2）、丙型流感病毒血凝素酯酶融合糖蛋白（HEF 1）的酯酶结构域、大肠杆菌硫酯酶I（TAP）的拓扑图进行了比较。大肠杆菌，来自念珠藻属的假定蛋白a1 r1529，而假设的YxiM前体都属于SGNH家族，这可能表明这些蛋白质可能来自一个共同的祖先。
YesT, a putative protein from Bacillus subtilis ATCC 6633 that has been Provisionally classified as a rhamnogalacturonan acetyl esterase (RGAE) in CE-12 family, was cloned, expressed in Escherichia coli Rosetta (DE3), and purified. The enzyme is monomeric with a molecular mass of 37 kDa and presents thermophilic properties similar to RGAE from Aspergillus aculeatus, although YesT is more alkaliphilic. The study of inhibitors confirmed the importance of the His and the nucleophilic Ser for the esterase activity, apart from the Asp from the catalytic triad. This enzyme also presents broad substrate specificity, and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, YesT achieves a synergistic effect together with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The primary sequence analysis and multiple sequence alignment revealed the lack of a two beta-stranded antiparallel sheet, which results in a clear change in the structure together with the disappearance of one of the three 3(10)-helices presented in RGAE structure. The similarities found in this article among the topological diagrams of RGAE, YesT, and Esterase A from Streptomyces scabies, Platelet-Activating Factor AcetylHydrolase, isoform Ib, alpha subunit [PAF-AH(Ib)alpha(1)], PAF-AH(Ib)alpha(2), the esterase domain from hemagglutinin esterase fusion glycoprotein (HEF1) from Influenza C virus, the thioesterase I (TAP) from E. coli, the hypothetical protein a1r1529 from Nostoc sp., and the hypothetical YxiM precursor that all belong to the SGNH family could indicate a possible divergence of such proteins from a common ancestor.