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首页> 外文期刊>Journal of Molecular Biology >Heterosubunit composition and crystal structures of a novel bacterial M16B metallopeptidase.
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Heterosubunit composition and crystal structures of a novel bacterial M16B metallopeptidase.

机译：新型细菌M16B金属肽酶的杂亚基组成和晶体结构。

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摘要

Three subfamilies of metallopeptidase family M16 enzymes--M16A, M16B, and M16C--are widely distributed among eukaryotes and prokaryotes. SPH2681, a periplasmic M16B protein found in Sphingomonas sp. strain A1, contains an HXXEH motif essential for Zn(2+) binding and catalytic activity. SPH2682 is another member of M16B, which lacks the metal-binding motif but conserves an active-site R/Y pair commonly found in the C-terminal half of M16 enzymes. Two genes coding for SPH2681 and SPH2682 assemble into a single operon in the bacterial genome. This study determined SPH2681 to be constitutively expressed in strain A1 cells grown on different carbon sources, suggesting a more general cellular function. SPH2681 and SPH2681/SPH2682 were overexpressed in Escherichia coli, purified, and characterized. SPH2681 was found to associate with SPH2682, forming a heterosubunit enzyme with peptidase activity, while SPH2681 alone exhibited no enzymatic activity. X-ray crystallography of the SPH2681/SPH2682 complex revealed two conformations (open and closed heterodimeric forms) within the same crystal. Compared with the closed form, the open form contains two subunits rotated away from each other by approximately 8 degrees , increasing the distance between the zinc ion and active-site residues by up to 8 A. In addition, many hydrogen bonds are formed or broken on change between the conformations of the heterodimers, suggesting that subunit dynamics is a prerequisite for catalysis. To our knowledge, this is the first report on both conformational forms of the same M16 peptidase, providing a unique insight into the general proteolytic mechanism of M16 proteases.
机译：金属肽酶家族M16酶的三个亚家族-M16A，M16B和M16C-在真核生物和原核生物中广泛分布。 SPH2681，一种在鞘氨醇单胞菌属种中发现的周质M16B蛋白。菌株A1，包含一个HXXEH主题，对Zn（2+）的结合和催化活性至关重要。 SPH2682是M16B的另一个成员，它缺少金属结合基序，但保留了通常在M16酶C末端一半中发现的活性位点R / Y对。编码SPH2681和SPH2682的两个基因在细菌基因组中组装成一个操纵子。这项研究确定SPH2681在不同碳源上生长的A1株细胞中组成性表达，提示其细胞功能更为普遍。 SPH2681和SPH2681 / SPH2682在大肠杆菌中过表达，纯化和鉴定。发现SPH2681与SPH2682缔合，形成具有肽酶活性的异亚基酶，而单独的SPH2681没有显示酶活性。 SPH2681 / SPH2682配合物的X射线晶体学分析显示同一晶体内有两个构象（开放和封闭的异源二聚体形式）。与封闭形式相比，开放形式包含两个彼此旋转大约8度的亚基，从而使锌离子和活性位残基之间的距离增加了多达8A。此外，许多氢键形成或断裂关于异二聚体的构象之间的变化，表明亚基动力学是催化的先决条件。据我们所知，这是关于同一M16肽酶两种构象形式的首次报道，提供了对M16蛋白酶一般蛋白水解机制的独特见解。

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