Vuillemin Marlene, Muschiol Jan, Zhang Yan, Holck Jesper, Barrett Kristian, Preben Morth Jens, Meyer Anne S, Zeuner Birgitte
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 221, 2800, Kgs. Lyngby, Denmark.
Current address: Ocean EcoSystems Biology Unit, Marine Ecology Research Department, GEOMAR Helmholtz Centre for Ocean Research, Wischhofstraβe 1-3, 24148, Kiel, Germany.
Chembiochem. 2025 Jan 14;26(2):e202400710. doi: 10.1002/cbic.202400710. Epub 2024 Oct 29.
The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.
糖苷水解酶家族20（GH20）主要以N-乙酰己糖胺酶（EC 3.2.1.52）为特征，已知的乳糖-N-生物糖苷酶（EC 3.2.1.140；LNBases）很少。LNBases催化乳糖-N-四糖（LNT）的降解，LNT是人乳寡糖的一个重要成分，从而支持健康的婴儿肠道微生物群发育。我们研究了GH20的多样性，以发现能释放二糖如乳糖-N-二糖（LNB）的新酶。我们的方法结合了肽聚类、序列分析和三维结构模型评估，以评估活性位点拓扑结构，重点关注亚位点-2的存在。五种LNBases对对硝基苯基-LNB有活性，四种对LNT有活性。一种酶对对硝基苯基-N-乙酰乳糖胺和对硝基苯基-LNB都有活性，这是N-乙酰乳糖胺酶（LacNAcase）活性的首次报道。对这个酶簇的探索导致鉴定出另外四种具有这种双重底物特异性的酶。比较特定LNBase（TrpyGH20）的确定晶体结构和具有双重LacNAcase/LNBase活性的酶的第一个晶体结构（TrdeGH20）发现，GH20酶有一个高度保守的亚位点-1，而-2亚位点有显著差异。TrdeGH20的亚位点-2更宽，能容纳与亚位点-1中的GlcNAc以β1,4-和β1,3-键连接的Gal。这些酶的生物技术应用可能包括复杂碳水化合物的结构解析和糖工程。
