Nyffenegger Christian, Nordvang Rune Thorbjørn, Jers Carsten, Meyer Anne S, Mikkelsen Jørn Dalgaard
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
PLoS One. 2017 Feb 3;12(2):e0171585. doi: 10.1371/journal.pone.0171585. eCollection 2017.
A sialidase (EC 3.2.1.18) from the non-pathogenic Trypanosoma rangeli, TrSA, has been shown to exert trans-sialidase activity after mutation of five specific amino acids in the active site (M96V, A98P, S120Y, G249Y, Q284P) to form the so-called TrSA5mut enzyme. By computational and hypothesis driven approaches additional mutations enhancing the trans-sialidase activity have been suggested. In the present work, we made a systematic combination of these mutations leading to seven new variants of the T. rangeli sialidase, having 6-16 targeted amino acid mutations. The resulting enzyme variants were analyzed via kinetics for their ability to carry out trans-sialidase reaction using CGMP and D-lactose as substrates. The sialidase variants with 15 and 16 mutations, respectively, exhibited significantly improved trans-sialidase activity for D-lactose sialylation. Our results corroborate, that computational studies of trans-glycosylation can be a valuable input in the design of novel trans-glycosidases, but also highlight the importance of experimental validation in order to assess the performance. In conclusion, two of the seven mutants displayed a dramatic switch in specificity from hydrolysis towards trans-sialylation and constitute the most potent trans-sialidase mutants of TrSA described in literature to date.
来自非致病性克氏锥虫的唾液酸酶（EC 3.2.1.18），即TrSA，已被证明在活性位点的五个特定氨基酸（M96V、A98P、S120Y、G249Y、Q284P）发生突变后会发挥转唾液酸酶活性，从而形成所谓的TrSA5mut酶。通过计算和假设驱动的方法，人们提出了其他增强转唾液酸酶活性的突变。在本研究中，我们对这些突变进行了系统组合，得到了七种新的克氏锥虫唾液酸酶变体，它们有6 - 16个靶向氨基酸突变。使用CGMP和D - 乳糖作为底物，通过动力学分析所得酶变体进行转唾液酸酶反应的能力。分别具有15个和16个突变的唾液酸酶变体对D - 乳糖唾液酸化表现出显著提高的转唾液酸酶活性。我们的结果证实，转糖基化的计算研究可以为新型转糖苷酶的设计提供有价值的参考，但也突出了实验验证对于评估性能的重要性。总之，七个突变体中的两个表现出从水解向转唾液酸化的特异性急剧转变，是迄今为止文献中描述的最有效的TrSA转唾液酸酶突变体。
