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首页> 外文期刊>Biochemistry >Major Change in Regiospecificity for the Exo-1,3-β-glucanase from Candida albicans following Its Conversion to a Glycosynthase
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Major Change in Regiospecificity for the Exo-1,3-β-glucanase from Candida albicans following Its Conversion to a Glycosynthase

机译：念珠菌的Exo-1,3-β-葡聚糖酶转化成糖合酶后区域特异性的重大变化

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The exo-1,3-β-glucanase (Exg) from Candida albicans is involved in cell wall β-D-glucan metabolism and morphogenesis through its hydrolase and transglycosidase activities. Previous work has shown that both these activities strongly favor β-1,3-linkages. The E292S Exg variant displayed modest glycosynthase activity using α-D-glucopyranosyl fluoride (α-GlcF) as the donor and pNP-β-D-glucopyranoside (pNPGlc) as the acceptor but surprisingly showed a marked preference for synthesizing β-1,6-linked over β-1,3- and β-1,4-linked disaccharide products. With pNPXyl as the acceptor, the preference became β-1,4 over β-1,3. The crystal structure of the glycosynthase bound to both of its substrates, α-GlcF and pNPGlc, is the first such ternary complex structure to be determined. The results revealed that the donor bound in the ?1 subsite, as expected, while the acceptor was oriented in the +1 subsite to facilitate β-1,6-linkage, thereby supporting the results from solution studies. A second crystal structure containing the major product of glycosynthesis, pNP-gentiobiose, showed that the ?1 subsite allows another docking position for the terminal sugar; i.e., one position is set up for catalysis, whereas the other is an intermediate stage prior to the displacement of water from the active site by the incoming sugar hydroxyls. The +1 subsite, an aromatic “clamp”, permits several different sugar positions and orientations, including a 180° flip that explains the observed variable regiospecificity. The pnitrophenyl group on the acceptor most likely influences the unexpectedly observed β-1,6-specificity through its interaction with F229. These results demonstrate that tailoring the specificity of a particular glycosynthase depends not only on the chemical structure of the acceptor but also on understanding the structural basis of the promiscuity of the native enzyme.
机译：白色念珠菌的exo-1,3-β-葡聚糖酶（Exg）通过其水解酶和转糖苷酶活性参与细胞壁β-D-葡聚糖的代谢和形态发生。先前的研究表明，这两种活性都强烈支持β-1,3-链接。 E292S Exg变体使用α-D-吡喃葡萄糖基氟（α-GlcF）作为供体和pNP-β-D-吡喃葡萄糖苷（pNPGlc）作为受体，显示出适度的糖合酶活性，但出乎意料地显示出显着的偏好合成β-1,6在β-1,3-和β-1,4-连接的二糖产物上连接。用pNPXyl作为受体，相对于β-1,3，偏好变为β-1,4。糖合酶与其两个底物α-GlcF和pNPGlc结合的晶体结构是首先确定的这种三元复合结构。结果表明，供体如预期的那样结合在β1亚位，而受体在+1亚位取向以促进β-1，6-连接，从而支持溶液研究的结果。包含糖合成的主要产物pNP-龙胆二糖的第二晶体结构表明，α1亚位点为末端糖提供了另一个对接位置。即，一个位置被设置用于催化，而另一个位置是在进入的糖羟基将水从活性位点置换之前的中间阶段。 +1子位点是芳族的“钳位”，允许几个不同的糖位置和方向，包括180°翻转，解释了观察到的可变区域特异性。受体上的对硝基苯基基团很可能通过与F229相互作用而影响到意外观察到的β-1,6-特异性。这些结果表明，定制特定糖合酶的特异性不仅取决于受体的化学结构，而且取决于对天然酶混杂的结构基础的理解。

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