Nordvang Rune T, Nyffenegger Christian, Holck Jesper, Jers Carsten, Zeuner Birgitte, Sundekilde Ulrik K, Meyer Anne S, Mikkelsen Jørn D
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
Department of Food Science, Aarhus University, Årslev, Denmark.
PLoS One. 2016 Jul 1;11(7):e0158434. doi: 10.1371/journal.pone.0158434. eCollection 2016.
Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.
唾液酸酶（3.2.1.18）如果其活性位点拓扑结构与克氏锥虫转唾液酸酶（EC 2.4.1.-）的活性位点拓扑结构一致，可能会表现出转唾液酸酶活性，以催化乳糖的唾液酸化。本研究旨在验证这样一个假设：克氏锥虫转唾液酸酶活性位点附近的两个特定氨基酸形成的特定芳香族三明治结构赋予了转唾液酸酶活性。在此基础上，通过对GenBank中2909种天然唾液酸酶进行迭代比对，鉴定出四种具有推定转唾液酸酶活性的酶，并在大肠杆菌中进行克隆和表达。其中，一种来源于副猪嗜血杆菌的酶SialH，在面向催化位点末端的蛋白质表面具有芳香族三明治结构（苯丙氨酸168；色氨酸366），并且确实被发现具有转唾液酸酶活性。SialH以酪蛋白糖巨肽为唾液酸供体、乳糖为受体，催化生成人乳寡糖3'-唾液酸乳糖以及新型转唾液酸化产物3-唾液酸乳糖。这些发现证实，克氏锥虫转唾液酸酶中的酪氨酸119和色氨酸312是赋予乳糖唾液酸化转唾液酸酶活性的芳香族三明治结构的一部分。通过合理的活性位点拓扑比对在计算机上鉴定转糖苷酶活性，因此被证明是在一大类糖基水解酶中筛选推定转唾液酸酶的快速工具。此外，该方法还提供了有助于理解转唾液酸酶结构-功能关系的数据。
