Probing the acceptor substrate binding site of Trypanosoma cruzi trans-sialidase with systematically modified substrates and glycoside libraries

Harrison, J.A.; Kartha, K. P. Ravindranathan; Fournier, E; Lowary, Todd L.; Malet, Carles; Nilsson, Ulf J.; Hindsgaul, Ole; Schenkman, Sérgio; Naismith, James H.; Field, Robert A.

doi:10.1039/c0ob00826e

Cited by 31 publications

(16 citation statements)

References 69 publications

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“…3). Interestingly, these results are in agreement with the recent finding that Gal(␤1-O(CH 2 ) 7 CH 3 ) and Gal(␤1-4)GlcNAc(␤1-O(CH 2 ) 7 CH 3 , modified at Gal C-2 or C-4 (H, F, OCH 3 , NH 2 ), greatly diminished or even prevented sialylation with TcTS and ␣-Neu5Ac pnitrophenyl glycoside as the donor, while those modified at Gal C-6 (H, F, OCH 3 , and NH 2 ) were well tolerated as acceptor substrates (25). NMR analysis of sialylated ␤3=-GL, ␤4=-GL, and ␤6=-GL.…”

Section: All Three Fractions Showed a Pseudomolecular Ion [M-h]mentioning

confidence: 99%

“…Due to its importance in host cell invasion and pathogenicity, TcTS has attracted substantial medical attention, and great strides have been made to discover inhibitors that may serve as therapeutics against the parasite (1,25). The unique sialic acid-transferring capabilities of TcTS also have been noticed by glycobiologists and biochemists and, using mainly 2-O-(4-methylumbelliferyl)-␣-Nacetylneuraminic acid (4MU-Neu5Ac), 2-O-(p-nitrophenyl)-␣-N-acetylneuraminic acid (pNP-Neu5Ac), and Neu5Ac(␣2-3)lactose (3=-SL) as donor substrates, the enzyme has been widely exploited in the chemo-enzymatic synthesis of various sialylated glycoconjugates (1).…”

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confidence: 99%

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Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

Wilbrink

Kate

Leeuwen

et al. 2014

Appl Environ Microbiol

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c trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(␣2-3)Gal(␤1-x)-glycans (sialo-glycans) to Gal(␤1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal) n Glc oligosaccharide mixtures (GOS) using bovine -casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3=-galactosyl-lactose (␤3=-GL), 4=-galactosyl-lactose (␤4=-GL), and 6=-galactosyl-lactose (␤6=-GL). This prebiotic GOS is prepared from lactose by incubation with suitable ␤-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (␣2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (␣2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of ␤3=-GL, ␤4=-GL, and ␤6=-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated ␤3=-GL and ␤4=-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of ␤6=-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the V max and k cat values were higher in the case of lactose.

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Section: All Three Fractions Showed a Pseudomolecular Ion [M-h]mentioning

confidence: 99%

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confidence: 99%

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

Wilbrink

Kate

Leeuwen

et al. 2014

Appl Environ Microbiol

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“…There is industrial interest in α‐rhamnosidases for use in the debittering of citrus juices and for the release of flavonoids from rhamnosylated precursors; in wine production they play a role in the hydrolysis of glycosylated terpene aroma compounds . In the context of expanding and diversifying the suite of enzymes available to us for carbohydrate biotransformations, we were drawn to consider α‐rhamnosidases.…”

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confidence: 99%

Crystal structure of a novel two domain GH78 family α‐rhamnosidase from Klebsiella oxytoca with rhamnose bound

et al. 2015

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The crystal structure of the GH78 family a-rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non-proline cis-peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays a-rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function.

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“…In order to assess which part of the galactose molecule may be important for trans-sialidase recognition, we set about probing the acceptor substrate binding site of the enzyme with systematically modified substrates and glycoside libraries (32). These compounds were based on modified octyl galactosides (33), a range of compounds derived from N-acetyllactosamine O-cyanomethyl ethers (34,35), and a >100 component library of thiogalactoside-derived compounds (36,37).…”

Section: Carbohydrate Librariesmentioning

confidence: 99%

Synthetic Glycans, Glycoarrays, and Glyconanoparticles To Investigate Host Infection byTrypanosoma cruzi

Field

Andrade

Campo

et al. 2011

ACS Symposium Series

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Carbohydrate libraries, synthetic glycans and glycopeptides are contributing to understanding of the role of sialic acid recognition in host infection by the parasite Trypanosoma cruzi. Further, glycoarray approaches are allowing us to explore similarities and differences in the carbohydrate-binding specificity of host lectins while glyconanoparticles enable us to ask questions about the structure, sialylation and recognition of parasite mucins.

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Probing the acceptor substrate binding site of Trypanosoma cruzi trans-sialidase with systematically modified substrates and glycoside libraries

Abstract: Trypanosoma cruzi trans-sialidase is a versatile catalyst for enzymatic α-2,3-sialylation reactions.

Cited by 31 publications

References 69 publications

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

Crystal structure of a novel two domain GH78 family α‐rhamnosidase from Klebsiella oxytoca with rhamnose bound

Synthetic Glycans, Glycoarrays, and Glyconanoparticles To Investigate Host Infection byTrypanosoma cruzi

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