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首页> 外文OA文献 >Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP 5 2-K from Arabidopsis thaliana
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Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP 5 2-K from Arabidopsis thaliana

机译：拟南芥InsP 5 2-K载脂蛋白形式的表达，纯化，结晶和初步X射线衍射分析

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摘要

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP 5 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP 6) from inositol 1,3,4,5,6-pentakisphos-phate (IP 5) and ATP. The first structure of IP 5 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP 5 or the product IP 6, and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP5 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP 5 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P2 12 12, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken.
机译：肌醇1,3,4,5,6-戊基磷酸2激酶（IP 5 2-K）是一种关键酶，它催化肌醇1,3,4,5,6-pentakisphos合成植酸（IP 6） -磷酸盐（IP 5）和ATP。 IP 5 2-K的第一个结构，来自拟南芥，先前已经得到解决。它仅在肌醇（底物IP 5或产物IP 6）存在下结晶，而在其游离状态（无肌醇）结晶失败。基于结构分析，已产生IP5 2-K（W129A）的点突变，以克服此限制并获得有关底物结合后蛋白质构象变化的信息。在此，描述了W129A IP 5 2-K的游离态和结合核苷酸的产生和结晶。这些晶体不同于天然晶体，属于正交晶空间群P2 12 12，单位晶格参数a = 66.00，b = 68.23，c = 105.80Å，a = 63.06，b = 71.80，c = 100.23Å 。晶体通过同步加速器辐射衍射至2.22Å（apo）和2.05Å（与核苷酸结合）的分辨率，每个不对称单元包含一个分子。已经使用分子置换方法确定了结构，并且正在进行改进。

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