Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis
Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis
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DOI：
10.1016/j.phytochem.2011.10.008
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发表时间：
2012-02-01
期刊：
影响因子：
3.8
通讯作者：
Brzobohaty, Bretislav
中科院分区：
文献类型：
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作者：
Filipi, Tomas;Mazura, Pavel;Brzobohaty, Bretislav
玉米β-D-葡萄糖苷酶Zm-p60.1以其储存/运输的形式释放活性细胞分裂素，其在烟草中的过度表达扰乱了玉米素的代谢。我们探索了活性位点微环境在微调Zm-p60.1底物特异性中的作用，特别是在W373K突变体中，使用定点随机突变来研究373位附近氨基酸变化的影响。获得了两个三重突变体(P372T/W373K/M376L和P372S/W373K/M376L)和三个双突变体(P372T/W373K、P372S/W373K和W373K/M376L)。它们在两种人工底物上的催化参数显示出底物催化作用与蛋白质结构之间的密切相关性。P372T/W373K/M376L对天然底物专一性的影响最显著：顺式-玉米素-O-β-D-吡喃葡萄糖苷的水解率与反式-玉米素-O-β-D-吡喃葡萄糖苷的比例从野生型的1.3转变为9.4，有利于顺式异构体。P372T/W373K/M376L的P372T和M376L突变也显著恢复了W373K突变株对顺式玉米素-O-β-D-吡喃葡萄糖苷的降解速度，最高可恢复野生型的60%。这些发现揭示了调节底物特异性的氨基酸残基之间的复杂关系，并表明了定点随机突变在改变和/或微调酶方面的有效性。特定异构体结合物的优先切割和操纵这种偏好的能力将允许开发用于详细探测和微调植物细胞分裂素新陈代谢的强大工具。(C)2011爱思唯尔有限公司。保留所有权利。
The maize beta-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-beta-D-glucopyranoside versus the trans-zeatin-O-beta-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-beta-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta. (C) 2011 Elsevier Ltd. All rights reserved.