Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity

Christian Nyffenegger, Rune Thorbjørn Nordvang, Carsten Jers, Anne S. Meyer, Jørn Dalgaard Mikkelsen

Research output: Contribution to journalJournal articleResearchpeer-review

275 Downloads (Pure)

Abstract

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.
Original languageEnglish
Article numbere0171585
JournalPLOS ONE
Volume12
Issue number2
Number of pages11
ISSN1932-6203
DOIs
Publication statusPublished - 2017

Bibliographical note

Copyright: © 2017 Nyffenegger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Fingerprint Dive into the research topics of 'Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity'. Together they form a unique fingerprint.

Cite this