Bioorganic studies on plant glycosidases by using p-glycosylamidines as research tools

以对糖基脒为研究工具的植物糖苷酶生物有机研究

• 批准号: 16310152
• 负责人: HIRATAKE Jun
• 金额: $ 10.11万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16310152/
• 关键词: β-glycosylamidines; β-glycosidase inhibitor; substrate analogues; glycon substrate specificity; plant glycosidases; affinity chromatography; diglycoside-specific glycosidases; X-ray crystal structure analysis; 基質アナログリガンド; 二糖配糖体認識機構; 静電的相互作用; アフ

The function and the physiological roles of plant β-glycosidases have been studied by using p-glycosylamidines as molecular probes. p-Glycosylamidines, newly developed p-glycosidase inhibitors that selectively inhibitβ-glycosidases according to their glycon substrate specificities, have been successfully used as ligand for affinity chromatography of glycosidases. High fidelities of β-glycosylamidines as specific binders toward theβ-glycosidases with the corresponding glycon substrate specificities enabled one-step affinity purification of specific β-glycosidases from natural samples according to their glycon substrate specificities as a sole marker. This method has been successfully used for the isolation of diglycoside-specific p-glycosidases from microbes and a p-N-acetylhexosaminidase from insect cells. The latter glycosidase is a key biosynthetic enzyme in processing insect-cell-specific N-glycans. With this research tool in hands, we have purified and characterized diglycoside-spe … More cific β-glycosidases (diglycosidases) from plants. The diglycosidases are thought to play a key role in self-defense system in plants by emitting defense chemicals such as linalool, 2-phenylethanol and mandelonitrile by cleaving their precursor diglycosides. β-Primeverosidase (PD), a typical diglycosidase found in tea leaves, has been purified to homogeneity by a newly developed pH-controlled affinity chromatography with p-primeverosylamidline as an affinity ligand. The enzymatic properties of PD and the binding mechanism of the β-primeverosylamidine inhibitor have been studied extensively. Furthermore, a gene encoding PD was cloned from tea leaves and was analyzed to find that the diglycosidases were a member of family 1 glycoside hydrolase with significant sequence similarities to the β-glucosidases in this family. This result suggests that plant diglycosidases have been evolved from family 1 β-glucosidases for cleavage of specific diglycosides and serve as a key enzyme in plant self-defense system. The recombinant PD was expressed and highly purified by the affinity chromatography for X-ray structural analysis. The high resolution X-ray crystal structure (1.8 Å) of PD in complex with the β-primeverosylamidine inhibitor revealed the amino acid residues recognizing the β-xylosyl moiety of β-primeveroside and the unique binding mode of β-primeverosylamidine ligand in the active site. These results cogently suggested the structural basis for their unique substrate specificities and the evolutional link between plant diglycosidases and family 1 p-glucosidases. Less

以对糖基胺为分子探针，研究了植物β糖苷酶的功能和生理作用。P-糖基胺是一种新开发的糖苷酶抑制剂，它根据糖基底物的特异性选择性地抑制β-糖苷酶，已成功地作为糖苷酶亲和层析的配基。β-糖胺作为β-糖苷酶的高保真度与相应的糖基底物特异性结合，使得根据其糖基底物特异性从天然样品中一步亲和纯化特定的β-糖苷酶成为唯一标记。该方法已成功地用于从微生物中分离二糖苷特异性的p-糖苷酶和从昆虫细胞中分离出一种p-N-乙酰己糖苷酶。后者是加工昆虫细胞特异性N-糖链的关键生物合成酶。利用这一研究工具，我们纯化并鉴定了二糖苷类化合物…植物中更特异的β-糖苷酶(二糖苷酶)。二糖苷酶被认为在植物的自我防御系统中发挥关键作用，它通过裂解其前体二糖苷而释放出芳樟醇、2-苯乙醇和扁桃腈等防御化学物质。β-Primeverosidase(PD)是一种存在于茶叶中的典型二糖苷酶，用p-Primeverosylamidline作为亲和配基，用最新发展起来的pH控制亲和层析对其进行了纯化。Pd的酶学性质和β-Primeverosylamidine抑制剂的结合机理已被广泛研究。此外，从茶叶中克隆了一个编码PD的基因，并对其进行了分析，发现二葡萄糖苷酶是糖苷水解酶家族1的成员，与该家族中的β-葡萄糖苷酶具有显著的序列相似性。这一结果表明，植物二糖苷酶是从家族1β-葡萄糖苷酶进化而来的，用于裂解特定的二糖苷类化合物，是植物自我防御系统中的关键酶。用亲和层析法对重组PD进行了高效表达和纯化，并进行了X射线结构分析。高分辨X射线晶体结构(1.8Å)揭示了Pd与β-Primeverosylamid抑制剂形成的络合物中识别β-Primeveroside的β-木糖基的氨基酸残基，以及活性部位β-Primeverosylamine配体独特的结合方式。这些结果有力地表明了它们独特底物专一性的结构基础，以及植物二糖苷酶和β-葡萄糖苷酶家族1之间的进化联系。较少