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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis, HPLC, and real-time ESI-MS.
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Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis, HPLC, and real-time ESI-MS.

机译：苏铁属的Cycas revoluta的V类几丁质酶催化的糖基转移反应：一项涉及定点诱变，HPLC和实时ESI-MS的研究。

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摘要

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.
机译：苏铁属苏铁（Cycas revoluta）的V类几丁质酶（CrChi-A）是发现具有转糖基化活性的第一种植物几丁质酶。为了确定引起转糖基化活性的结构决定簇，我们突变了两个芳香残基Phe166和Trp197，它们很可能位于受体结合位点，并对突变的酶（F166A，W197A）进行了表征。当通过HPLC监测针对几丁质寡糖的酶促反应的时间过程时，比活性降低至野生型的比活性的约5-10％，并且由于单个突变而显着减少了转糖基化产物的量。通过比较HPLC和实时ESI-MS获得的反应时间过程，我们发现转糖基化反应发生在HPLC所用的条件下，而不是ESI-MS条件下。用于HPLC测定的较高的底物浓度（5 mM）可能导致几丁质酶催化的转糖基化。通过HPLC获得的时间过程的动力学分析表明，与野生型相比，两种突变酶中+1亚位点的糖残基亲和力均大大降低。实时ESI-MS测定的抑制剂异蒜素的IC（50）值不受单个突变的影响很大，表明异蒜素结合位点的状态（从-3到-1个亚位点）在突变中没有变化。突变的酶。我们得出结论，CrChi-A中Phe166和Trp197的芳香族侧链参与了糖基转移受体的结合，从而控制了酶的糖基转移活性。

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