1. Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity.
- Author
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Nyffenegger C, Nordvang RT, Jers C, Meyer AS, and Mikkelsen JD
- Subjects
- Catalytic Domain, Glycoproteins genetics, Glycosylation, Kinetics, Lactose metabolism, Mutation genetics, Neuraminidase genetics, Glycoproteins metabolism, Neuraminidase metabolism, Trypanosoma rangeli enzymology
- 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., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
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