Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in <i>Escherichia coli</i> K1 bacteriophages

Jakobsson, Elina; Jokilammi, Anne; Aalto, Juha; Ollikka, Pauli; Lehtonen, Jukka V.; Hirvonen, Harri; Finne, Jukka

doi:10.1042/bj20070177

Cited by 32 publications

(36 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has also been shown that endoNF preferentially binds sialic acid oligomers in this helical form (85), and the enzyme could provide the second acid or base catalyst, normally required in a glycosidase mechanism, to further increase the rate of depolymerization. This is consistent with the observation that amino acid changes around the putative active site of endo-sialidases drastically change the catalytic efficiency, by locally distorting the polymer in a way that does not promote transition state formation but does not affect binding of the polymer, because there are multiple sugar-binding sites (48).…”

Section: Discussionsupporting

confidence: 77%

“…This mutant was shown to be properly folded and capable of binding substrate but showed no sialidase activity toward the sialotrioside substrate under a range of conditions. This is not entirely unexpected because recent studies on other endo-sialidases have shown that other mutations around the putative active site can have a drastic effect on catalysis without affecting binding (48). The complete ablation of sialidase activity, however, is in apparent contrast to previous studies of the E581A mutant, in which a 5% residual sialidase activity was observed (15).…”

Section: Discussioncontrasting

confidence: 54%

See 1 more Smart Citation

A New Sialidase Mechanism

Morley

Willis

Whitfield

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 77%

Section: Discussioncontrasting

confidence: 54%

A New Sialidase Mechanism

Morley

Willis

Whitfield

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Five endosialidase genes have been cloned from different E. coli K1 bacteriophages (7)(8)(9)(10)(11)(12)(13). The proteins encoded by these genes share a common architecture with three linearly organized domains as follows.…”

mentioning

confidence: 99%

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

Schwarzer

Stummeyer

Haselhorst

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Endosialidases (endoNs), as identified so far, are tailspike proteins of bacteriophages that specifically bind and degrade the ␣2,8-linked polysialic acid (polySia) capsules of their hosts. The crystal structure solved for the catalytic domain of endoN from coliphage K1F (endoNF) revealed a functional trimer. Folding of the catalytic trimer is mediated by an intramolecular C-terminal chaperone domain. Release of the chaperone from the folded protein confers kinetic stability to endoNF. In mutant c(S), the replacement of serine 911 by alanine prevents proteolysis and generates an enzyme that varies in activity from wild type. Using soluble polySia as substrate a 3-times higher activity was detected while evaluation with immobilized polySia revealed a 190-fold reduced activity. Importantly, activity of c(S) did not differ from wild type with tetrameric sialic acid, the minimal endoNF substrate. Furthermore, we show that the presence of the chaperone domain in c(S) destabilizes binding to polySia in a similar way as did selective disruption of a polySia binding site in the stalk domain. The improved catalytic efficiency toward soluble polySia observed in these mutants can be explained by higher dissociation and association probabilities, whereas inversely, an impaired processivity was found. The fact that endoNF is a processive enzyme introduces a new molecular basis to explain capsule degradation by bacteriophages, which until now has been regarded as a result of cooperative interaction of tailspike proteins. Moreover, knowing that release of the chaperone domain confers kinetic stability and processivity, conservation of the proteolytic process can be explained by its importance in phage evolution.

show abstract

“…Yellow residues have been described to be directly involved in catalysis [106,127] whereas green amino acids have been mutated and identified as polysialic acid binding residues [12,106]. Blue colored residues have been identified in inactive endoNA or endoNE mutants [15,69] enzymatic activity [106]. Further residues were identified by natural mutation of the endosialidases from K1A and K1E to be important for catalysis (depicted in blue in Fig.…”

Section: The Active Site Of Endosialidasementioning

confidence: 98%

“…Further residues were identified by natural mutation of the endosialidases from K1A and K1E to be important for catalysis (depicted in blue in Fig. 3c; [69], see Sect. 12.1).…”

Section: The Active Site Of Endosialidasementioning

confidence: 99%

Endosialidases: Versatile Tools for the Study of Polysialic Acid

Jakobsson

Schwarzer

Jokilammi

et al. 2012

Topics in Current Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Cited by 32 publications

References 44 publications

A New Sialidase Mechanism

A New Sialidase Mechanism

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

Endosialidases: Versatile Tools for the Study of Polysialic Acid

Contact Info

Product

Resources

About