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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

Identification of the novel trans-sialylation product by MALDI-TOF.MALDI-TOF of anion exchange purified SialH reaction mixture, i.e. all sialylated compounds in the reaction mixture, before and after β-galactosidase treatment. MALDI-TOF/TOF of m/z 632 in the untreated sample is consistent with the presence of a SL type compound. MALDI-TOF/TOF of m/z 470 ion in the β-galactosidase treated sample is the result of sialyllactose breakdown, which suggests a non-sialylated galactose moiety and thus, a sialylated glucose moiety. MS-MS of the m/z 470 ion is consistent with breakdown of sialylated glucose. Two ions resulted in significant peaks (BSA = sialic acid-ion and Aglc = glucose-ion) and are, together with the suggested bond cleavage, indicated in the figure.
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pone.0158434.g005: Identification of the novel trans-sialylation product by MALDI-TOF.MALDI-TOF of anion exchange purified SialH reaction mixture, i.e. all sialylated compounds in the reaction mixture, before and after β-galactosidase treatment. MALDI-TOF/TOF of m/z 632 in the untreated sample is consistent with the presence of a SL type compound. MALDI-TOF/TOF of m/z 470 ion in the β-galactosidase treated sample is the result of sialyllactose breakdown, which suggests a non-sialylated galactose moiety and thus, a sialylated glucose moiety. MS-MS of the m/z 470 ion is consistent with breakdown of sialylated glucose. Two ions resulted in significant peaks (BSA = sialic acid-ion and Aglc = glucose-ion) and are, together with the suggested bond cleavage, indicated in the figure.

Mentions: The four identified candidate enzymes were examined for trans-sialidase activity upon successful expression in E. coli as His6-tagged proteins. As previously, casein glycomacropeptide (CGMP) was used as donor substrate and lactose as the acceptor substrate for 3’SL production [16, 28, 31] and this reaction was performed in a time study setup, to evaluate trans-sialidase activity (Table 4; Fig 4). CGMP is a side stream product from cheese production and contains 9 mM bound α-2,3- and α-2,6-linked sialic acid in a ratio of 1:1 [32], which makes it an interesting substrate for industrial production of sialylated compounds. Our primary candidate, SialH, was capable of catalyzing trans-sialylation with a product-to-hydrolysis ratio peaking at above 2.5 for the production of 3’SL, making it the first bacterial trans-sialidase to be identified (Table 4). This novel trans-sialidase produced a relatively stable 3’SL concentration of 1.5 mM (Fig 4). Interestingly, another reaction product was formed besides 3’SL and SA. Analyzed by MALDI-TOF, the product gave rise to an m/z 632-ion suggesting that it is an SL compound (Fig 5), but HPAEC-PAD analysis ruled out that it was 6´SL (S2 Fig). The compound was susceptible to β-galactosidase hydrolysis, indicating that the compound was not sialylated on the Gal moiety at the non-reducing end (S2 Fig). The lack of sialylation at the non-reducing galactose was confirmed by NMR (S3 Fig), and the novel compound was identified as 3-sialyllactose (3SL). 3’-sialylated glycans are the primary product of trans-sialylation reactions using the Trypanosoma trans-sialidases, but also 6’-sialylation has been reported using other sialidases [30]. So far, 3SL is a novel trans-sialylation product.


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)

Identification of the novel trans-sialylation product by MALDI-TOF.MALDI-TOF of anion exchange purified SialH reaction mixture, i.e. all sialylated compounds in the reaction mixture, before and after β-galactosidase treatment. MALDI-TOF/TOF of m/z 632 in the untreated sample is consistent with the presence of a SL type compound. MALDI-TOF/TOF of m/z 470 ion in the β-galactosidase treated sample is the result of sialyllactose breakdown, which suggests a non-sialylated galactose moiety and thus, a sialylated glucose moiety. MS-MS of the m/z 470 ion is consistent with breakdown of sialylated glucose. Two ions resulted in significant peaks (BSA = sialic acid-ion and Aglc = glucose-ion) and are, together with the suggested bond cleavage, indicated in the figure.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0158434.g005: Identification of the novel trans-sialylation product by MALDI-TOF.MALDI-TOF of anion exchange purified SialH reaction mixture, i.e. all sialylated compounds in the reaction mixture, before and after β-galactosidase treatment. MALDI-TOF/TOF of m/z 632 in the untreated sample is consistent with the presence of a SL type compound. MALDI-TOF/TOF of m/z 470 ion in the β-galactosidase treated sample is the result of sialyllactose breakdown, which suggests a non-sialylated galactose moiety and thus, a sialylated glucose moiety. MS-MS of the m/z 470 ion is consistent with breakdown of sialylated glucose. Two ions resulted in significant peaks (BSA = sialic acid-ion and Aglc = glucose-ion) and are, together with the suggested bond cleavage, indicated in the figure.
Mentions: The four identified candidate enzymes were examined for trans-sialidase activity upon successful expression in E. coli as His6-tagged proteins. As previously, casein glycomacropeptide (CGMP) was used as donor substrate and lactose as the acceptor substrate for 3’SL production [16, 28, 31] and this reaction was performed in a time study setup, to evaluate trans-sialidase activity (Table 4; Fig 4). CGMP is a side stream product from cheese production and contains 9 mM bound α-2,3- and α-2,6-linked sialic acid in a ratio of 1:1 [32], which makes it an interesting substrate for industrial production of sialylated compounds. Our primary candidate, SialH, was capable of catalyzing trans-sialylation with a product-to-hydrolysis ratio peaking at above 2.5 for the production of 3’SL, making it the first bacterial trans-sialidase to be identified (Table 4). This novel trans-sialidase produced a relatively stable 3’SL concentration of 1.5 mM (Fig 4). Interestingly, another reaction product was formed besides 3’SL and SA. Analyzed by MALDI-TOF, the product gave rise to an m/z 632-ion suggesting that it is an SL compound (Fig 5), but HPAEC-PAD analysis ruled out that it was 6´SL (S2 Fig). The compound was susceptible to β-galactosidase hydrolysis, indicating that the compound was not sialylated on the Gal moiety at the non-reducing end (S2 Fig). The lack of sialylation at the non-reducing galactose was confirmed by NMR (S3 Fig), and the novel compound was identified as 3-sialyllactose (3SL). 3’-sialylated glycans are the primary product of trans-sialylation reactions using the Trypanosoma trans-sialidases, but also 6’-sialylation has been reported using other sialidases [30]. So far, 3SL is a novel trans-sialylation product.

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