title: Rational design of a new Trypanosoma rangeli trans-sialidase for efficient sialylation of glycans.
abstract: This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197-203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197-203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197-203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Jers
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Malwina
authors.initials: M
authors.lastname: Michalak
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Dorte M
authors.initials: DM
authors.lastname: Larsen
authors.affiliation: Department of Chemistry, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Kasper P
authors.initials: KP
authors.lastname: Kepp
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern University of Denmark, Odense, Denmark.
authors.forename: Haiying
authors.initials: H
authors.lastname: Li
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Yao
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern University of Denmark, Odense, Denmark.
authors.forename: Finn
authors.initials: F
authors.lastname: Kirpekar
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D016415:Sequence Alignment; D013379:Substrate Specificity; D057509:Trypanosoma rangeli / Q000235:genetics / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D009439:Neuraminidase