The Hydrolase and Transferase Activity of an Inverting Mutant Sialidase Using Non-natural β-Sialoside Substrates

Watson, Jacqueline N.; Indurugalla, Deepani; Cheng, Lydia; Narine, and Arun A.; Bennet, Andrew J.

doi:10.1021/bi061489x

Cited by 11 publications

(25 citation statements)

References 34 publications

(81 reference statements)

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“…The only previous reports of inverting sialidases are those describing mutant enzymes, wherein the catalytic tyrosine nucleophile of a retaining sialidase has been mutated to an alanine (70,75,76). Removing the nucleophilic catalyst resulted in a switch from a retaining mechanism in the wild type sialidase to an inverting mechanism in the mutant.…”

Section: Discussionmentioning

confidence: 99%

A New Sialidase Mechanism

Morley

Willis

Whitfield

et al. 2009

Journal of Biological Chemistry

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Bacteriophages specific for Escherichia coli K1 express a tailspike protein that degrades the polysialic acid coat of E. coli K1 that is essential for bacteriophage infection. This enzyme is specific for polysialic acid and is a member of a family of endosialidases. This family is unusual because all other previously reported sialidases outside of this family are exo-or trans-sialidases. The recently determined structure of an endo-sialidase derived from bacteriophage K1F (endoNF) revealed an active site that lacks a number of the residues that are conserved in other sialidases, implying a new, endo-sialidase-specific catalytic mechanism. Using synthetic trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for hydrolysis at a single cleavage site were determined. Measurement of k cat /K m at a series of pH values revealed a dependence on a single protonated group of pK a 5. Mutation of a putative active site acidic residue, E581A, resulted in complete loss of sialidase activity. Direct 1 H NMR analysis of the hydrolysis of trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting sialidase. All other wild type sialidases previously reported are retaining glycosidases, implying a new mechanism of sialidase action specific to this family of endo-sialidases.The important role played by sialylated glycoconjugates (1) in both homeostasis (2, 3) and disease states (4 -7) has led to the extensive study of the enzymes responsible for the addition and removal of sialic acid. With the exception of the ␤-linked CMP donor sugar (8), naturally occurring sialic acid glycosides are found in the ␣-configuration, their syntheses being catalyzed by sialyltransferases. All of the sialyltransferases are thus inverting enzymes (9). The corresponding wild type sialidases (both exoand trans-sialidases) that have been studied all share a very similar set of active site residues and cleave the terminal ␣-linked sialic acid residue by the same catalytic mechanism (10 -13). This conserved active site includes a tyrosine acting as the nucleophilic catalyst, two aspartate and/or glutamate residues as the general acid/base, and a trio of arginines associated with the sialic acid carboxylate. Hydrolysis occurs via an acid/ base-catalyzed double-displacement mechanism involving a covalent sialyl-enzyme intermediate, resulting in overall retention of configuration at the anomeric center.A noteworthy exception to this mechanism comes from the sialidases of family 6 which are the only class of enzymes found to hydrolyze within a sialic acid polymer (14) (endo-sialidase) because all others reported cleave only the terminal sialic acid residue (exo-sialidase). The x-ray crystal structure of a member of this family, derived from an Escherichia coli K1 bacteriophage, was recently solved revealing a putative active site that is similar in geometry to those of exo-sialidases but missing the tyrosine nucleophilic catalyst, one of the two acid catalysts, and one of the three arginines (15). This stark contrast in...

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Section: Discussionmentioning

confidence: 99%

A New Sialidase Mechanism

Morley

Willis

Whitfield

et al. 2009

Journal of Biological Chemistry

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“…Sialidases employ a nucleophilic Tyr instead of the more common Asp/Glu. Not being actual glycosynthases, mutations of this nucleophilic Tyr in Micromonospora viridifaciens sialidase (EC 3.2.1.18; GH 33) resulted in a number of mutants that retained hydrolytic activity with either retention or inversion of configuration. , Using phenyl β- d -sialic acid as a donor, the inverting mutant Y370G catalyzed production of sialyl-lactose with a yield of 13%, with >90% being the α(2,6)-isomer and with no subsequent product hydrolysis …”

Section: Protein Engineeringmentioning

confidence: 99%

“…111,112 Using phenyl β-D-sialic acid as a donor, the inverting mutant Y370G catalyzed production of sialyl-lactose with a yield of 13%, with >90% being the α(2,6)-isomer and with no subsequent product hydrolysis. 113 However, it follows that glycosynthase catalysis releases either hydrofluoric acid (HF) or hydrazoic acid (HN 3 ) as its byproduct. Although numerous methods for the removal of fluoride ions from aqueous solution exist, 114 this issue of removal has hardly been addressed by any of the otherwise thorough reviews (see, e.g., refs 103, 105, and 106) of glycosynthases and the use of glycosyl fluorides as substrates.…”

Section: ■ Protein Engineeringmentioning

confidence: 99%

Methods for Improving Enzymatic Trans-glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics

Zeuner

Jers

Mikkelsen

et al. 2014

J. Agric. Food Chem.

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Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote "reverse" catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-l-fucosidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.

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“…Despite the observed success with the use of S-glycosides as inhibitors of glycosidases including sialidases (Watson et al, 2006) the Neu5Acα2-3-S-Galβ- O -octyl was found to show no significant inhibition of TcTS even at millimolar concentrations (Harrison et al, 2001). The low inhibition rates observed by the Neu5Acα2-3-S-Galβ- O -octyl suggest that the conformations acquired by this compound in solution might not be recognized by the TcTS.…”

Section: Inhibition Of Sialic Acid Transference By Tctsmentioning

confidence: 99%

Sialic acid: a sweet swing between mammalian host and Trypanosoma cruzi

Freire-de-Lima¹,

Oliveira²,

Neves³

et al. 2012

Front. Immun.

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Commonly found at the outermost ends of complex carbohydrates in extracellular medium or on outer cell membranes, sialic acids play important roles in a myriad of biological processes. Mammals synthesize sialic acid through a complex pathway, but Trypanosoma cruzi, the agent of Chagas’ disease, evolved to obtain sialic acid from its host through a trans-sialidase (TcTS) reaction. Studies of the parasite cell surface architecture and biochemistry indicate that a unique system comprising sialoglycoproteins and sialyl-binding proteins assists the parasite in several functions including parasite survival, infectivity, and host–cell recognition. Additionally, TcTS activity is capable of extensively remodeling host cell glycomolecules, playing a role as virulence factor. This review presents the state of the art of parasite sialobiology, highlighting how the interplay between host and parasite sialic acid helps the pathogen to evade host defense mechanisms and ensure lifetime host parasitism.

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The Hydrolase and Transferase Activity of an Inverting Mutant Sialidase Using Non-natural β-Sialoside Substrates

Cited by 11 publications

References 34 publications

A New Sialidase Mechanism

A New Sialidase Mechanism

Methods for Improving Enzymatic Trans-glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics

Sialic acid: a sweet swing between mammalian host and Trypanosoma cruzi

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