Department of Bioorganic Chemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Department of Medical Biochemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Chemistry. 2018 Jul 11;24(39):9983-9992. doi: 10.1002/chem.201801902. Epub 2018 Jun 21.
N-Glycans direct protein function, stability, folding and targeting, and influence immunogenicity. While most glycosidases that process N-glycans cleave a single sugar residue at a time, enzymes from glycoside hydrolase family 99 are endo-acting enzymes that cleave within complex N-glycans. Eukaryotic Golgi endo-1,2-α-mannosidase cleaves glucose-substituted mannose within immature glucosylated high-mannose N-glycans in the secretory pathway. Certain bacteria within the human gut microbiota produce endo-1,2-α-mannanase, which cleaves related structures within fungal mannan, as part of nutrient acquisition. An unconventional mechanism of catalysis was proposed for enzymes of this family, hinted at by crystal structures of imino/azasugars complexed within the active site. Based on this mechanism, we developed the synthesis of two glycosides bearing a spiro-epoxide at C-2 as electrophilic trap, to covalently bind a mechanistically important, conserved GH99 catalytic residue. The spiro-epoxyglycosides are equipped with a fluorescent tag, and following incubation with recombinant enzyme, allow concentration, time and pH dependent visualization of the bound enzyme using gel electrophoresis.
N-聚糖可直接影响蛋白质的功能、稳定性、折叠和靶向，并影响免疫原性。虽然大多数加工 N-聚糖的糖苷酶一次只能切割一个糖残基，但糖苷水解酶家族 99 的酶是内切酶，可在复杂的 N-聚糖内切割。真核高尔基体内切 1,2-α-甘露糖苷酶在分泌途径中切割未成熟的葡萄糖化高甘露糖 N-聚糖内的葡萄糖取代甘露糖。人类肠道微生物群中的某些细菌产生内切 1,2-α-甘露聚糖酶，作为获取营养的一部分，它可切割真菌甘露聚糖中相关结构。该家族的酶提出了一种非常规的催化机制，晶体结构暗示了活性位点内与亚氨基/氮杂糖复合物。基于该机制，我们开发了两种带有 C-2 螺环环氧的糖苷的合成，作为亲电陷阱，与具有重要机械意义的保守 GH99 催化残基共价结合。螺环环氧糖苷带有荧光标记，与重组酶孵育后，可使用凝胶电泳在浓度、时间和 pH 依赖性条件下可视化结合的酶。
