- A practical synthesis of capped 4-methylumbelliferyl hyaluronan disaccharides and tetrasaccharides as potential hyaluronidase substrates
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The synthesis of hyaluronan dimers and tetramers equipped with a 4-methylumbelliferyl group at the reducing end to potentially allow monitoring of hyaluronidase activities is described. The 4-OH at the non-reducing glucuronate in the presented series is either removed or methylated to prohibit transglycosylase reactions, leading to a total of four probes.
- Gold, Henrik,Munneke, Stefan,Dinkelaar, Jasper,Overkleeft, Herman S.,Aerts, Johannes M.F.G.,Codée, Jeroen D.C.,Van Der Marel, Gijs A.
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p. 1467 - 1478
(2011/08/22)
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- Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method
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In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher kcat and 15 fold-lower Km than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-β-D-N- N′-N″-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of β-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)2 (main product), GlcNAc, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.
- Synstad, Bjornar,Vaaje-Kolstad, Gustav,Cederkvist, F. Henning,Saua, Silje F.,Horn, Svein J.,Eijsink, Vincent G. H.,Sorlie, Morten
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p. 715 - 723
(2008/09/19)
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- Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: Mechanistic and structural insights into inhibitor selectivity and transition state poise
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O-GlcNAcase catalyzes the cleavage of β-O-linked 2-acetamido-2-deoxy- β-D-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2′-methyl-α-D-glucopyranoso[2,1-d]-Δ2′- thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal β-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.
- Whitworth, Garrett E.,Macauley, Matthew S.,Stubbs, Keith A.,Dennis, Rebecca J.,Taylor, Edward J.,Davies, Gideon J.,Greig, Ian R.,Vocadlo, David J.
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p. 635 - 644
(2007/10/03)
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