Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.

甘露糖苷酶是一组多样化的糖苷水解酶，在寡甘露糖聚糖、糖缀合物和糖蛋白的甘露糖修剪中起关键作用，这些过程涉及许多细胞过程，例如聚糖生物合成和代谢、结构调节、细胞识别和细胞-病原体相互作用。外甘露糖苷酶和内甘露糖苷酶切割聚糖中甘露糖苷键的特定糖苷键，可用于基于酶的方法对异构聚糖结构进行测序。来自Xanthomonas manihotis的 α1-6-甘露糖苷酶被称为高度特异性的外切糖苷酶，可从寡糖中去除未分支的 α1-6 连接的甘露糖残基。然而，我们通过使用酶促反应、高效阴离子交换色谱和液相色谱质谱测序，发现这种 α1-6-甘露糖苷酶在处理支链杂合和复杂聚糖时也具有意想不到的 β1-4-半乳糖苷酶活性。 . 我们对 α1-6-甘露糖苷酶与聚糖底物的对接模拟揭示了相对较浅的口袋中潜在的相互作用残基，与糖苷水解酶 125 家族中的同源酶略有不同，这可能是观察到底物结合和随后末端的更高混杂性的原因聚糖水解。对 α1-6-甘露糖苷酶的新型 β1-4-半乳糖苷酶活性的观察提供了独特的见解，了解其对无支链聚糖末端 α1-6-甘露糖和杂合体末端 β1-4-半乳糖的底物结构依赖性加工的双功能活性和复杂的聚糖。因此，该发现表明这种 α1-6-甘露糖苷酶的双重糖苷酶特异性以及在用于聚糖异构体的结构解析时需要仔细考虑。