Use of conformationally restricted pyridinium α-D-<i>N</i>-acetylneuraminides to probe specificity in bacterial and viral sialidases

Watson, Jacqueline N.; Knoll, Tara L.; Chen, Johnny H; Chou, Doug T. H.; Borgford, Thor J.; Bennet, Andrew J.

doi:10.1139/o04-126

Cited by 6 publications

(4 citation statements)

References 39 publications

(52 reference statements)

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“…Of note, the M. Viridifaciens sialidase efficiently processes a broad range of substrate structures. Specifically, the CP for the hydrolysis of 3 (Table 3) is between 60-and 1200fold greater than those reported for the enzyme-catalyzed hydrolysis of pyridinium R-D-N-acetylneuraminide by a series of bacterial and viral sialidases (33).…”

Section: Discussionmentioning

confidence: 66%

Structure and Mechanism of Action of an Inverting Mutant Sialidase

et al. 2005

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Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Brønsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure 3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the active site. First, the anomeric oxygen atom of the hydrolysis product (beta-N-acetylneuraminic acid) and four water molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310 moves to make a strong hydrogen bond to one of the bound water molecules.

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Section: Discussionmentioning

confidence: 66%

Structure and Mechanism of Action of an Inverting Mutant Sialidase

et al. 2005

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“…The recently published mechanism for sialidase-catalyzed hydrolyses (Scheme ), which is a refinement of previously advocated schemes, ,− is based on a comprehensive series of KIE measurements on the kinetic parameter V max for the Micromonospora viridifaciens enzyme . In detail, the bound sugar residue in the accumulating Michaelis complex is in a skew boat conformation (likely the 6 S 2 ) with subsequent glycosidic bond cleavage occurring to give a 1 C 4 chair intermediate that is covalently bound to the active site tyrosine residue .…”

Section: Resultsmentioning

confidence: 99%

Transition State Analysis of Vibrio cholerae Sialidase-Catalyzed Hydrolyses of Natural Substrate Analogues

et al. 2012

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A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-α-d-neuraminyl-(2→3)-β-d-galactopyranosyl-(1→4)-1-thio-β-d-glucopyranoside; Neu5Acα2,3LacβSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacβSPh for the ring oxygen ((18)V/K), leaving group oxygen ((18)V/K), C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6βSPh for C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacβSPh and Neu5Acα2,6LacβSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening ((4)H(5) half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6βSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.

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“…3b,c). The Salmonella typhimurium (ST) sialidase NanH was selected for its relatively high K M value (mM range) against polyvalent targets [31][32][33] , enhancement of NK cell-mediated ADCC, and stability (Supplementary Fig. 3b-d).…”

Section: Minimizing Off-target Sialidase Activitymentioning

confidence: 99%

Targeted Desialylation Overcomes Glyco-Immune Checkpoints and Potentiates the Anticancer Immune Response in Vivo

Gray¹,

Stanczak²,

Han³

et al. 2019

Preprint

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<div><div><div><p>Currently approved immune checkpoint inhibitor (ICI) therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, the majority of patients across cancer types still fail to respond. Addressing alternative pathways that mediate immune suppression could enhance ICI efficacy. One such mechanism is the increase in sialic acid-containing proteins and lipids (sialoglycans) in malignancy, which recently has been shown to inhibit immune cell activation through multiple mechanisms including Siglec receptor binding, and therefore represents a targetable glyco-immune checkpoint. Here, we report the design of a trastuzumab- sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells in vivo. In a syngeneic orthotopic HER2+ breast cancer model, targeted desialylation delayed tumor growth and enhanced immune cell infiltration and activation, leading to prolonged survival of mice with trastuzumab-resistant breast cancer. Thus, antibody-sialidase conjugates represent a promising modality for cancer immune therapy.</p></div></div></div>

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Use of conformationally restricted pyridinium α-D-N-acetylneuraminides to probe specificity in bacterial and viral sialidases

Cited by 6 publications

References 39 publications

Structure and Mechanism of Action of an Inverting Mutant Sialidase

Structure and Mechanism of Action of an Inverting Mutant Sialidase

Transition State Analysis of Vibrio cholerae Sialidase-Catalyzed Hydrolyses of Natural Substrate Analogues

Targeted Desialylation Overcomes Glyco-Immune Checkpoints and Potentiates the Anticancer Immune Response in Vivo

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