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 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.
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
Christian
C
Nyffenegger
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
Rune Thorbjørn
RT
Nordvang
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
Carsten
http://orcid.org/0000-0002-9930-3137
C
Jers
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
Anne S
AS
Meyer
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
Jørn Dalgaard
JD
Mikkelsen
D020134:Catalytic Domain; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D057509:Trypanosoma rangeli / Q000201:enzymology*
D006023:Glycoproteins; C069715:trans-sialidase; D009439:Neuraminidase; D007785:Lactose