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It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Nordvang RT, Nyffenegger C, Holck J, Jers C, Zeuner B, Sundekilde UK, Meyer AS, Mikkelsen JD - PLoS ONE (2016)

Bottom Line: SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor.The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases.The approach moreover provided data that help understand structure-function relations of trans-sialidases.

View Article: PubMed Central - PubMed

Affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.

ABSTRACT
Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

No MeSH data available.


Related in: MedlinePlus

The active site of the trans-sialidase from Trypanosoma cruzi (TcTS).The 3’-sialyllactose substrate (yellow) is shown positioned in the active site of TcTS. The active site residues (top 9 residues in Table 1) bind the sialic acid moiety of the substrate by a number of hydrogen bonds (A), whereas the aromatic sandwich (W312 and Y119) aligns to the lactose moiety and binds it in the tight cleft above the active site (B). An overview of the interactions between enzyme and substrate is given in Table 1. The model is based on PDB entry 1S0I [11].
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pone.0158434.g001: The active site of the trans-sialidase from Trypanosoma cruzi (TcTS).The 3’-sialyllactose substrate (yellow) is shown positioned in the active site of TcTS. The active site residues (top 9 residues in Table 1) bind the sialic acid moiety of the substrate by a number of hydrogen bonds (A), whereas the aromatic sandwich (W312 and Y119) aligns to the lactose moiety and binds it in the tight cleft above the active site (B). An overview of the interactions between enzyme and substrate is given in Table 1. The model is based on PDB entry 1S0I [11].

Mentions: A visual representation of the TcTS Michaelis complex with 3’SL is presented in Fig 1 and an overview of the 11 residues interacting with the substrate in the Michaelis complex is given in Table 1 [9, 11]. Of these, R35, R245, and R314 form an Arg triad responsible for sialyl carboxylate fixation, whereas D59 is the general acid/base catalyst and Y342 acts as the nucleophile; all of these are conserved in all sialidases and trans-sialidases [9, 24]. In addition, R53, D96, W120, and Q195 also take part in sialyl fixation, while W312 and Y119 interacts with and orient the acceptor (Table 1; Fig 1). Aiming to identify new trans-sialidases, this study focused on the residues interacting in the Michaelis complex of TcTS (Fig 1; Table 1), which are conserved in all trypanosomal trans-sialidases, rather than looking for the seven amino acid residues conserved in all hydrolytic sialidases, which apart from the Arg triad, the general acid/base catalyst, and the nucleophile also include E230 acting as a base catalyst for the nucleophile and E357 stabilizing R35 through a salt bridge [9, 24]. Several factors pointed to the aromatic sandwich comprised of W312 and Y119, which are unique to trypanosomal trans-sialidases, as a good trans-sialidase identification marker. Apart from being reported to be essential for the trans-sialidase activity of TcTS, W312 in the aromatic sandwich is located close to R314, which is one of the conserved residues in all sialidases and trans-sialidases [9, 11]. The close proximity of the two residues enabled the creation of a search motif which could be used for a database search, namely ΩxRDR, where ‘Ω’ represents an aromatic amino acid and ‘x’ is any residue (Box 1).


It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Nordvang RT, Nyffenegger C, Holck J, Jers C, Zeuner B, Sundekilde UK, Meyer AS, Mikkelsen JD - PLoS ONE (2016)

The active site of the trans-sialidase from Trypanosoma cruzi (TcTS).The 3’-sialyllactose substrate (yellow) is shown positioned in the active site of TcTS. The active site residues (top 9 residues in Table 1) bind the sialic acid moiety of the substrate by a number of hydrogen bonds (A), whereas the aromatic sandwich (W312 and Y119) aligns to the lactose moiety and binds it in the tight cleft above the active site (B). An overview of the interactions between enzyme and substrate is given in Table 1. The model is based on PDB entry 1S0I [11].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930215&req=5

pone.0158434.g001: The active site of the trans-sialidase from Trypanosoma cruzi (TcTS).The 3’-sialyllactose substrate (yellow) is shown positioned in the active site of TcTS. The active site residues (top 9 residues in Table 1) bind the sialic acid moiety of the substrate by a number of hydrogen bonds (A), whereas the aromatic sandwich (W312 and Y119) aligns to the lactose moiety and binds it in the tight cleft above the active site (B). An overview of the interactions between enzyme and substrate is given in Table 1. The model is based on PDB entry 1S0I [11].
Mentions: A visual representation of the TcTS Michaelis complex with 3’SL is presented in Fig 1 and an overview of the 11 residues interacting with the substrate in the Michaelis complex is given in Table 1 [9, 11]. Of these, R35, R245, and R314 form an Arg triad responsible for sialyl carboxylate fixation, whereas D59 is the general acid/base catalyst and Y342 acts as the nucleophile; all of these are conserved in all sialidases and trans-sialidases [9, 24]. In addition, R53, D96, W120, and Q195 also take part in sialyl fixation, while W312 and Y119 interacts with and orient the acceptor (Table 1; Fig 1). Aiming to identify new trans-sialidases, this study focused on the residues interacting in the Michaelis complex of TcTS (Fig 1; Table 1), which are conserved in all trypanosomal trans-sialidases, rather than looking for the seven amino acid residues conserved in all hydrolytic sialidases, which apart from the Arg triad, the general acid/base catalyst, and the nucleophile also include E230 acting as a base catalyst for the nucleophile and E357 stabilizing R35 through a salt bridge [9, 24]. Several factors pointed to the aromatic sandwich comprised of W312 and Y119, which are unique to trypanosomal trans-sialidases, as a good trans-sialidase identification marker. Apart from being reported to be essential for the trans-sialidase activity of TcTS, W312 in the aromatic sandwich is located close to R314, which is one of the conserved residues in all sialidases and trans-sialidases [9, 11]. The close proximity of the two residues enabled the creation of a search motif which could be used for a database search, namely ΩxRDR, where ‘Ω’ represents an aromatic amino acid and ‘x’ is any residue (Box 1).

Bottom Line: SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor.The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases.The approach moreover provided data that help understand structure-function relations of trans-sialidases.

View Article: PubMed Central - PubMed

Affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.

ABSTRACT
Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

No MeSH data available.


Related in: MedlinePlus