Limits...
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

Homology modeling of active sites of the four selected trans-sialidase candidates.The four enzyme candidates selected for in vitro testing, SialH (green), SialA (red), SialM (blue), and SialP (orange), were modeled by homology modeling using TcTS as the template (gray). The active site and aromatic sandwich residues (indicated in TcTS with arrows in the SialH figure) are shown. The four candidates were selected based on two criteria: 1) restoration of the active site with its nine sialyl interacting residues, and 2) the formation of a narrow (closed) binding cleft above the active site, preferably with an aromatic sandwich composed of two aromatic amino acid residues. Whereas all of the selected candidates were predicted to have sterically closed active sites, SialH was the only candidate predicted to display an aromatic sandwich. The aromatic sandwich of SialH is comprised of W366 and F168 and aligns (in the model) almost perfectly to the one found in TcTS.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4930215&req=5

pone.0158434.g002: Homology modeling of active sites of the four selected trans-sialidase candidates.The four enzyme candidates selected for in vitro testing, SialH (green), SialA (red), SialM (blue), and SialP (orange), were modeled by homology modeling using TcTS as the template (gray). The active site and aromatic sandwich residues (indicated in TcTS with arrows in the SialH figure) are shown. The four candidates were selected based on two criteria: 1) restoration of the active site with its nine sialyl interacting residues, and 2) the formation of a narrow (closed) binding cleft above the active site, preferably with an aromatic sandwich composed of two aromatic amino acid residues. Whereas all of the selected candidates were predicted to have sterically closed active sites, SialH was the only candidate predicted to display an aromatic sandwich. The aromatic sandwich of SialH is comprised of W366 and F168 and aligns (in the model) almost perfectly to the one found in TcTS.

Mentions: For two of the 16 sequences, no template for homology modeling was available (S2 Table). Homology models for the other 14 candidates were constructed and were–without regard to the quality of the models–aligned to TcTS for inspection (Fig 2; S1 Fig). Of these, 12 sequences fell for criterion 1 as they did not contain the nine sialyl interacting active site residues (S1 Fig; S2 Table). In an effort to identify any sequences that might have been missed during the initial sequence search, the two remaining sequences of the enzyme candidate models were submitted to an NCBI BLAST search and the two closest homologues were inspected to identify sequences with an aromatic residue in a position equivalent to W312 in TcTS. This led to the identification of two such sequences that were introduced in the study, modeled, and successfully evaluated against the two selection criteria (S2 Table). In total, four candidates fulfilled the two criteria, i.e. they had all nine sialyl interacting active site residues, as well as a narrow binding cleft sandwich structure, namely (putative) sialidases from Actinomyces oris, Haemophilus parasuis, Manheimia heamolytica, and Pasteurella multocida, which were named SialA, SialH, SialM, and SialP, respectively (Table 2; Fig 2). SialM, SialP, and SialH had sequence identities to each other of 58–62%, whereas SialA had sequence identities to the three others of 25–29%.


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)

Homology modeling of active sites of the four selected trans-sialidase candidates.The four enzyme candidates selected for in vitro testing, SialH (green), SialA (red), SialM (blue), and SialP (orange), were modeled by homology modeling using TcTS as the template (gray). The active site and aromatic sandwich residues (indicated in TcTS with arrows in the SialH figure) are shown. The four candidates were selected based on two criteria: 1) restoration of the active site with its nine sialyl interacting residues, and 2) the formation of a narrow (closed) binding cleft above the active site, preferably with an aromatic sandwich composed of two aromatic amino acid residues. Whereas all of the selected candidates were predicted to have sterically closed active sites, SialH was the only candidate predicted to display an aromatic sandwich. The aromatic sandwich of SialH is comprised of W366 and F168 and aligns (in the model) almost perfectly to the one found in TcTS.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0158434.g002: Homology modeling of active sites of the four selected trans-sialidase candidates.The four enzyme candidates selected for in vitro testing, SialH (green), SialA (red), SialM (blue), and SialP (orange), were modeled by homology modeling using TcTS as the template (gray). The active site and aromatic sandwich residues (indicated in TcTS with arrows in the SialH figure) are shown. The four candidates were selected based on two criteria: 1) restoration of the active site with its nine sialyl interacting residues, and 2) the formation of a narrow (closed) binding cleft above the active site, preferably with an aromatic sandwich composed of two aromatic amino acid residues. Whereas all of the selected candidates were predicted to have sterically closed active sites, SialH was the only candidate predicted to display an aromatic sandwich. The aromatic sandwich of SialH is comprised of W366 and F168 and aligns (in the model) almost perfectly to the one found in TcTS.
Mentions: For two of the 16 sequences, no template for homology modeling was available (S2 Table). Homology models for the other 14 candidates were constructed and were–without regard to the quality of the models–aligned to TcTS for inspection (Fig 2; S1 Fig). Of these, 12 sequences fell for criterion 1 as they did not contain the nine sialyl interacting active site residues (S1 Fig; S2 Table). In an effort to identify any sequences that might have been missed during the initial sequence search, the two remaining sequences of the enzyme candidate models were submitted to an NCBI BLAST search and the two closest homologues were inspected to identify sequences with an aromatic residue in a position equivalent to W312 in TcTS. This led to the identification of two such sequences that were introduced in the study, modeled, and successfully evaluated against the two selection criteria (S2 Table). In total, four candidates fulfilled the two criteria, i.e. they had all nine sialyl interacting active site residues, as well as a narrow binding cleft sandwich structure, namely (putative) sialidases from Actinomyces oris, Haemophilus parasuis, Manheimia heamolytica, and Pasteurella multocida, which were named SialA, SialH, SialM, and SialP, respectively (Table 2; Fig 2). SialM, SialP, and SialH had sequence identities to each other of 58–62%, whereas SialA had sequence identities to the three others of 25–29%.

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