Proteolytic Release of the Intramolecular Chaperone Domain Confers Processivity to Endosialidase F

Schwarzer, David; Stummeyer, Katharina; Haselhorst, Thomas; Freiberger, Friedrich; Rode, Bastian; Grove, Melanie; Scheper, Thomas; Itzstein, Mark von; Mühlenhoff, Martina; Gerardy‐Schahn, Rita

doi:10.1074/jbc.m808475200

Cited by 28 publications

(60 citation statements)

References 41 publications

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“…Based upon these data, it appears that endoNF has at least three subsites on the nonreducing side of the cleavage site, and the minimum substrate requirement is for the Ϫ1, Ϫ2, and Ϫ3 subsites (64) to all be occupied. This observation is consistent with the cleavage patterns of other endo-sialidases that have been reported using simple oligosialoside substrates (53,65,66) and particularly with a very recent publication showing that the tetramer is the true minimum substrate (67). Our data also serve to confirm that the enzyme does not work via an exo-sialidase mode from the nonreducing terminus but rather via an endo-mode.…”

Section: Resultssupporting

confidence: 80%

“…The measured kinetic parameters are similar to those reported for simple oligosialosides (66,67), indicating that the coumarin modification does not significantly affect the binding of these modified oligosialosides to endoNF. The pH profile of endoNF shows that a single ionizable residue with a pK a of ϳ5 in either the free enzyme or free substrate is essential for catalysis, suggesting a carboxylic acid.…”

Section: Discussionsupporting

confidence: 74%

“…These values are similar to those obtained for other sialic acid oligomers (e.g. K m ϭ ϳ1.2 mM and k cat ϭ 72 min Ϫ1 for a degree of polymerization 10) (53) and closely correlate with the recent analysis of the degradation of a sialic acid tetramer by endoNF (67). Analysis of the enzyme stability at a series of pH values (Fig.…”

Section: Resultssupporting

confidence: 67%

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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: Resultssupporting

confidence: 80%

Section: Discussionsupporting

confidence: 74%

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A New Sialidase Mechanism

Morley

Willis

Whitfield

et al. 2009

Journal of Biological Chemistry

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“…This confers processivity to O-antigen hydrolysis. Processive TSP have also been found in phages infecting encapsulated bacteria (33). Here, processivity appears to result from secondary polysaccharide-binding sites on the same protein, although there is no evidence for secondary LPS-binding sites on P22 TSP.…”

Section: Discussionmentioning

confidence: 82%

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Andres¹,

Hanke

Baxa³

et al. 2010

Journal of Biological Chemistry

138

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Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.

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“…However, under in vitro conditions with more exhaustive digestion the minimum chain length of polysialic acid to be cleaved has ranged from trimer to octamer [103][104][105] (for an overview see [77]). Interestingly, endoNF is able to cleave oligosialic acid with a minimum of four residues whereas three residues are required at the non-reducing end [105]. In the crystal structure of the inactive mutant endoNF-H350A, a tri-sialic acid is bound to the active site.…”

Section: Specificity Of Endosialidasementioning

confidence: 99%

Endosialidases: Versatile Tools for the Study of Polysialic Acid

Jakobsson

Schwarzer

Jokilammi

et al. 2012

Topics in Current Chemistry

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Polysialic acid is an α2,8-linked N-acetylneuraminic acid polymer found on the surface of both bacterial and eukaryotic cells. Endosialidases are bacteriophage-borne glycosyl hydrolases that specifically cleave polysialic acid. The crystal structure of an endosialidase reveals a trimeric mushroom-shaped molecule which, in addition to the active site, harbors two additional polysialic acid binding sites. Folding of the protein crucially depends on an intramolecular C-terminal chaperone domain that is proteolytically released in an intramolecular reaction. Based on structural data and previous considerations, an updated catalytic mechanism is discussed. Endosialidases degrade polysialic acid in a processive mode of action, and a model for its mechanism is suggested. The review summarizes the structural and biochemical elucidations of the last decade and the importance of endosialidases in biochemical and medical applications. Active endosialidases are important tools in studies on the biological roles of polysialic acid, such as the pathogenesis of septicemia and meningitis by polysialic acid-encapsulated bacteria, or its role as a modulator of the adhesion and interactions of neural and other cells. Endosialidase mutants that have lost their polysialic acid cleaving activity while retaining their polysialic acid binding capability have been fused to green fluorescent protein to provide an efficient tool for the specific detection of polysialic acid.

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Proteolytic Release of the Intramolecular Chaperone Domain Confers Processivity to Endosialidase F

Cited by 28 publications

References 41 publications

A New Sialidase Mechanism

A New Sialidase Mechanism

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Endosialidases: Versatile Tools for the Study of Polysialic Acid

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