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首页> 外文期刊>The FEBS journal >Functional analysis of the aglycone-binding site of the maize beta-glucosidase Zm-p60.1
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Functional analysis of the aglycone-binding site of the maize beta-glucosidase Zm-p60.1

机译：玉米β-葡萄糖苷酶Zm-p60.1糖苷配基结合位点的功能分析

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摘要

beta-Glucosidases such as Zm-p60.1 (Zea mays) and Bgl4:1 (Brassica napus) have implicated roles in regulating plant development by releasing biologically active cytokinins from O-glucosides. A key determinant of substrate specificity in Zm-p60.1 is the F193-F200-W373-F461 cluster. However, despite sharing the same substrates, amino acids in the active sites of Zm-p60.1 and Bgl4:1 differ dramatically. In members of the Brassicaceae we found a group of beta-glucosidases sharing both high similarity to Bgl4:1 and a consensus motif A-K-K-L corresponding to the F193-F200-W373-F461 cluster. To study the mechanism of substrate specificity further, we generated and analyzed four single (F193A, F200K, W373K and F461L) and one quadruple (F193A-F200K-W373K-F461L) mutants of Zm-p60.1. The F193A mutant showed a specific increase in affinity for a small polar aglycone, and a deep decrease in k(cat) compared with the wild-type. Formation of a cavity with decreased hydrophobicity, and significant consequent alterations in ratios of reactive and non-reactive complexes, revealed by computer modeling, may explain the observed changes in kinetic parameters of the F193 mutant. The large decrease in k(cat) for the W373K mutant was unexpected, but the findings are consistent with the F193-aglycone-W373 interaction playing a dual role in the enzyme's catalytic action; influencing both substrate specificity, and the catalytic rate by fixing the glucosidic bond in a favorable orientation for attack by the catalytic pair. Investigation of the combined effects of all of the mutations in the quadruple mutant of Zm-p60.1 was precluded by extensive alterations in its structure and almost complete abolition of its enzymatic activity.
机译：诸如Zm-p60.1（Zea mays）和Bgl4：1（Brassica napus）等β-葡萄糖苷酶通过从O-葡萄糖苷释放生物活性细胞分裂素而参与调节植物发育。 Zm-p60.1中底物特异性的关键决定因素是F193-F200-W373-F461簇。然而，尽管共享相同的底物，Zm-p60.1和Bgl4：1的活性位点中的氨基酸却截然不同。在十字花科的成员中，我们发现了一组与Bgl4：1具有高度相似性且与F193-F200-W373-F461簇相对应的共有基序A-K-K-L共享高度相似的β-葡萄糖苷酶。为了进一步研究底物特异性的机制，我们生成并分析了Zm-p60.1的四个单个（F193A，F200K，W373K和F461L）和一个四个（F193A-F200K-W373K-F461L）突变体。与野生型相比，F193A突变体显示出对小的极性糖苷配基的亲和力有特定的增加，而k（cat）则有明显的降低。通过计算机建模揭示，疏水性降低的空腔的形成以及随之而来的反应性和非反应性复合物比率的显着变化可能解释了F193突变体动力学参数的变化。 W373K突变体的k（cat）大幅降低是出乎意料的，但发现与F193-糖苷配基-W373相互作用在酶的催化作用中起双重作用是一致的。通过将葡糖苷键固定在有利的方向上来影响底物特异性和催化速率，以防止被催化对攻击。 Zm-p60.1的四重突变体中所有突变的联合作用研究由于其结构的广泛改变和几乎完全取消其酶促活性而无法进行。

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