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

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-糖苷胺是一种新开发的p-糖苷酶抑制剂，根据其糖基底物的特异性选择性地抑制β-糖苷酶，已成功地用作糖苷酶亲和层析的配体。β-糖基氨基作为具有相应糖基底物特异性的β-糖苷酶的特异性结合物的高保真度使得根据其糖基底物特异性作为唯一标记从天然样品中一步亲和纯化特异性β-糖苷酶。该方法已成功地从微生物中分离出二糖苷特异性对糖苷酶和从昆虫细胞中分离出对n -乙酰己糖氨酸酶。后者糖苷酶是处理昆虫细胞特异性n -聚糖的关键生物合成酶。有了这个研究工具，我们已经从植物中纯化和表征了更具体的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，通过裂解前体二糖苷，释放出芳樟醇、2-苯乙醇和扁桃腈等防御化学物质。以对primeverosylamidline为亲和配体，采用ph控制亲和色谱法纯化了茶叶中典型的二糖苷酶β-Primeverosidase （PD）。人们对PD的酶学性质和β-primeverosylamidine抑制剂的结合机制进行了广泛的研究。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，发现该二糖苷酶是1家族糖苷水解酶的成员，与该家族的β-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1科β-葡萄糖苷酶进化而来的，用于切割特定的二糖苷，是植物自卫系统的关键酶。通过亲和层析对重组蛋白进行了表达和纯化，并进行了x射线结构分析。PD与β-primeverosylamidine抑制剂配合物的高分辨率x射线晶体结构（1.8 Å）揭示了识别β- primeverosylamid苷β-木基部分的氨基酸残基以及β-primeverosylamidine配体在活性位点的独特结合模式。这些结果有力地说明了它们独特底物特异性的结构基础，以及植物二糖苷酶与1家族p-葡萄糖苷酶之间的进化联系。少

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