Structural and Kinetic Analysis of Bacillus subtilis N-Acetylglucosaminidase Reveals a Unique Asp-His Dyad Mechanism

Litzinger, Silke; Fischer, Sebastian; Polzer, Patrick; Diederichs, Kay; Welte, Wolfram; Mayer, Christoph

doi:10.1074/jbc.m110.131037

Cited by 110 publications

(154 citation statements)

References 55 publications

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“…Furthermore, Asp 320 interacts closely (2.6 Å) with the N⑀1 of His 302 , suggesting these two residues may act in tandem to effect general acid catalysis and relay a proton from Asp 320 to the leaving group (Fig. 3B) (17). The LA residue is fully enclosed in the Ϫ1 subsite.…”

Section: Mutation Of Active Site Residues-the Predicted Catalytic Resmentioning

confidence: 99%

“…6). Indeed, the Asp 320 -His 302 dyad of BpGH117 is similar to the Asp 232 -His 324 catalytic dyad observed for family 3 glycoside hydrolase BsNagZ from B. subtilis (17). A notable difference between the mechanisms of these two enzymes, however, is that BsNagZ uses a retaining mechanism, and therefore the histidine residue acts as both acid and base, whereas the histidine in BpGH117 acts only as an acid in the proposed inverting mechanism of catalysis for this enzyme.…”

mentioning

confidence: 96%

“…This analysis reveals distortion of the constrained 3,6-anhydro-L-galactose residue to a conformation conducive to hydrolysis of the glycosidic bond. In the proposed inverting catalytic mechanism, a histidine residue is suggested to act as the general acid, which would be the first observation of its kind for a glycoside hydrolase and would add to the recent example of a histidine acting as the acid/base catalytic residue for the retaining catalytic mechanism used by the N-acetylglucosaminidase from Bacillus subtilis (17).…”

mentioning

confidence: 99%

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Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Hehemann

Smyth

Yadav

et al. 2012

Journal of Biological Chemistry

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Background:The catalytic mechanism and substrate recognition required for cleavage of the ␣-linkage in agarose are unclear.Results: Structural analysis of a family 117 glycoside hydrolase details substrate recognition and supports an inverting mechanism. Conclusion: GH117 enzymes use substrate distortion and an unusual general acid for catalysis. Significance: Microbes may utilize alternate strategies to catalyze the degradation of polysaccharides with unique structural characteristics.

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Section: Mutation Of Active Site Residues-the Predicted Catalytic Resmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

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Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Hehemann

Smyth

Yadav

et al. 2012

Journal of Biological Chemistry

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“…This residue is conserved in GH3 enzymes and is indeed suitably positioned within the BglP active site to serve as the catalytic nucleophile forming the key reaction intermediate. The active site contains most of the same residues identified in other GH3 enzymes 37 as being involved in substrate recognition, such as Lys193 and His194 of the signature sequence. Also consistent with other members of the GH3 subfamily to which BglP belongs, the imidazole of a histidine residue (His206), is positioned ~5.5 Å from the carboxylate of Asp288 on the opposite face of the hexose ring of the bound intermediate and is thus well positioned to assist in catalysis by acting as a general acid/base.…”

Section: As Seen Inmentioning

confidence: 99%

“…Errors were derived from the fit to the experimental data provided by GraphPad. 37 and Burkholderia cenocepacia (BcNagZ) (pink carbons) (PDB: 4GNV). The overlay reveals that Gln205 in BglP is replaced by Ser residues in the NagZ enzymes, enabling them to accommodate the C2 N-acetyl group of GlcNAc.…”

Section: Pre-steady State Kineticsmentioning

confidence: 99%

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Macdonald

Patel

Larmour

et al. 2018

Journal of Biological Chemistry

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Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-D-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenol from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to Nacetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. GH3 β-glycoside phosphorylase from a metagenomic library 2 Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.Carbohydrate active enzymes (CAZymes) are the biocatalysts responsible for the assembly, degradation and modification of glycans in biological systems 1 . They are also widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel and bioproduct development [2][3][4][5] . While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive starting materials, such as nucleotide phosphosugars 6 . The high-cost of these materials makes de novo industrial-scale glycan synthesis difficult and usually non-viable.One class of CAZyme that offers a potential solution to the high costs typically associated with enzymatic glycan synthesis is that of the glycoside phosphorylases (GPs), which are increasingly being recognized and used for the biocatalysis and biotransformation of glycans 7-9 . These enzymes ordinarily carry out phosphorolysis by transferring a glycosyl moiety from the nonreducing end of a di-or polysaccharide substrate onto inorganic phosphate, thereby generating a sugar-1-phosphate 10 ( Figure 1A). GPs distinguish themselves from most CAZymes in that the hydrolytic free energy associated with the glycosidic e...

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Glycosidases: Functions, Families and Folds

Kötzler

Hancock

Withers

2014

Encyclopedia of Life Sciences

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Based in part on the previous version of this eLS article ' Glycosidases: Functions, Families and Folds' (2007) by Susan M Hancock and Stephen G Withers.Glycosidases catalyse the hydrolysis of glycosidic linkages, thereby degrading oligosaccharides and glycoconjugates, the structurally most diverse class of biopolymers. These efficient and highly specific catalysts play important roles in biological processes thus a detailed knowledge of glycosidase function is invaluable for understanding and controlling diseases and for industrial applications. The classification of this huge class of enzymes into families on the basis of amino acid sequence has provided a highly valuable tool for the analysis of structure-function relationships. Furthermore, the steady increase in three-dimensional structural information is revealing further evolutionary relationships between glycosidase families. In addition to the majority of glycosidases that act via the classical Koshland mechanisms, a growing number of such enzymes that use unusual mechanisms are being uncovered. This confluence of bioinformatics, structural and mechanistic studies has greatly advanced glycosidase engineering and the development of specific glycosidase inhibitors.

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Structural and Kinetic Analysis of Bacillus subtilis N-Acetylglucosaminidase Reveals a Unique Asp-His Dyad Mechanism

Cited by 110 publications

References 55 publications

Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Glycosidases: Functions, Families and Folds

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