title: Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3'- and 6'-sialyllactose.
abstract: Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Yao
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. Electronic address: cj@kt.dtu.dk.
authors.forename: Carsten
authors.initials: C
authors.lastname: Jers
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Haiying
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Finn
authors.initials: F
authors.lastname: Kirpekar
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007785:Lactose / Q000031:analogs & derivatives / Q000096:biosynthesis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis; D016979:Pasteurella multocida / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein
chemical_list: C421467:3'-sialyllactose; C403777:6'-sialyllactose; D001426:Bacterial Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012799:Sialyltransferases; C069715:trans-sialidase; D009439:Neuraminidase; D007785:Lactose
keywords: Pasteurella multocida; Regioselectivity; Sialyllactose; Sialyltransferase; Site-directed mutagenesis; Trans-sialidase