Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity.
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...
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doaj-3094ebce845b40e1b7f18b6db8f16af62020-11-25T00:09:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01122e017158510.1371/journal.pone.0171585Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity.Christian NyffeneggerRune Thorbjørn NordvangCarsten JersAnne S MeyerJørn Dalgaard MikkelsenA 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.http://europepmc.org/articles/PMC5291517?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Christian Nyffenegger Rune Thorbjørn Nordvang Carsten Jers Anne S Meyer Jørn Dalgaard Mikkelsen |
spellingShingle |
Christian Nyffenegger Rune Thorbjørn Nordvang Carsten Jers Anne S Meyer Jørn Dalgaard Mikkelsen Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. PLoS ONE |
author_facet |
Christian Nyffenegger Rune Thorbjørn Nordvang Carsten Jers Anne S Meyer Jørn Dalgaard Mikkelsen |
author_sort |
Christian Nyffenegger |
title |
Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. |
title_short |
Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. |
title_full |
Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. |
title_fullStr |
Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. |
title_full_unstemmed |
Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. |
title_sort |
design of trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2017-01-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC5291517?pdf=render |
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