Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Jäger, Szilvia; Kiss, László

doi:10.1007/s11274-004-2609-2

Cited by 7 publications

(8 citation statements)

References 35 publications

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“…The activity of the Asp2863Ala mutation was too low for the K m to be accurately determined; thus, the apparent k cat was estimated. The decrease in the catalytic activity of this mutant of approximately 4 to 5 orders of magnitude (Table 3) coupled with the observation that this residue aligns with previously identified catalytic nucleophilic residues in GH3 enzymes (2,9,11,31,34,35,40) provides support for the assignment of Asp286 as the catalytic nucleophile for Xyl3B.…”

Section: Discussionsupporting

confidence: 80%

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B ₁ 4

et al. 2010

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Prevotella bryantii B 1 4 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded ␤-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k cat /K m for pNP-␤-Dxylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k cat /K m for pNP-␤-D-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between ␤-xylosides and ␤-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.Among the bacterial genera in the bovine rumen, Prevotella species are the most numerically abundant species by both culture counts and by analysis of 16S rRNA abundances (13,43). These organisms contribute to the breakdown of plant protein and hemicellulose within the rumen microbial ecosystem. Despite these important physiological roles, relatively little is known about the molecular mechanisms for plant polysaccharide utilization by ruminal Prevotella species. The genomes for Prevotella bryantii B 1 4 and P. ruminicola 23 have recently been sequenced and harbor a large number of putative glycoside hydrolase (GH) genes

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Section: Discussionsupporting

confidence: 80%

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B ₁ 4

et al. 2010

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“…Owing to these drawbacks, no active site residue could be identified using this inactivator. Finally, the N-bromoacetyl glycosylamines and bromoketone C-glycosides (represented by the general structures 3f-h) have been used to label and inactivate several glycosidases (Naider et al 1972;Black et al 1993;Keresztessy et al 1994;Tull et al 1996;Howard and Withers 1998a,b;Chir et al 2002;Kiss et al 2002;Vocadlo et al 2002;Jager and Kiss 2005). Unlike the classes of compounds 3a-e discussed above, the N-bromoacetyl glycosylamines and bromoketone C-glycosides have typically proven to be sufficiently stable toward spontaneous decomposition to be useful as labeling agents when care is taken to select the proper inactivator, as described below.…”

Section: Other Affinity Labelsmentioning

confidence: 99%

“…Interestingly, this is the same methionine as that which was labeled by the galactosylmethyl triazene described above (Sinnott andSmith 1976, 1978;Fowler et al 1978). In a second case in which this reagent was employed, the authors propose that the catalytic nucleophile is the site of labeling on the basis of the pH dependence on the rate of inactivation and the pH/rate profile of enzymatic substrate hydrolysis (Jager and Kiss 2005). However pH/rate profiles can be notoriously difficult to interpret (Knowles 1976), so in the absence of a structural study and a detailed kinetic analysis of both wild-type and site-directed mu-tants (see Vocadlo et al 2002 for a good example of this type of analysis), caution should be exercised.…”

Section: Other Affinity Labelsmentioning

confidence: 99%

Covalent inhibitors of glycosidases and their applications in biochemistry and biology

2008

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Glycoside hydrolases are important enzymes in a number of essential biological processes. Irreversible inhibitors of this class of enzyme have attracted interest as probes of both structure and function. In this review we discuss some of the compounds used to covalently modify glycosidases, their use in residue identification, structural and mechanistic investigations, and finally their applications, both in vitro and in vivo, to complex biological systems.

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“…13,44,77−79,81,83−85 However, the inactivation constants, k i , for other N-bromoacetylglycosylamine inhibitors are 2−3 orders of magnitude lower. 13,44,[77][78][79]81,83,85 The large error on the apparent K i value, in this case, reflects an apparently poor ability to saturate the enzyme and a correspondingly flat k app vs [I] curve (see Figure S2). Similarly, saturation inhibition kinetics were not achieved with the tetrasaccharide inhibitor GGG3G-β-NHCOCH 2 Br (compound 5; see Figure S3 in the Supporting Information).…”

Section: ■ Results and Discussionmentioning

confidence: 91%

Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases

et al. 2021

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Small molecule irreversible inhibitors are valuable tools for determining catalytically important active-site residues and revealing key details of the specificity, structure, and function of glycoside hydrolases (GHs). β-glucans that contain backbone β(1,3) linkages are widespread in nature, e.g., mixed-linkage β(1,3)/β(1,4)-glucans in the cell walls of higher plants and β(1,3)glucans in yeasts and algae. Commensurate with this ubiquity, a large diversity of mixed-linkage endoglucanases (MLGases, EC 3.2.1.73) and endo-β(1,3)-glucanases (laminarinases, EC 3.2.1.39 and EC 3.2.1.6) have evolved to specifically hydrolyze these polysaccharides, respectively, in environmental niches including the human gut. To facilitate biochemical and structural analysis of these GHs, with a focus on MLGases, we present here the facile chemo-enzymatic synthesis of a library of active-site-directed enzyme inhibitors based on mixed-linkage oligosaccharide scaffolds and N-bromoacetylglycosylamine or 2-fluoro-2-deoxyglycoside warheads. The effectiveness and irreversibility of these inhibitors were tested with exemplar MLGases and an endo-β(1,3)-glucanase. Notably, determination of inhibitor-bound crystal structures of a human-gut microbial MLGase from Glycoside Hydrolase Family 16 revealed the orthogonal labeling of the nucleophile and catalytic acid/base residues with homologous 2-fluoro-2-deoxyglycoside and N-bromoacetylglycosylamine inhibitors, respectively. We anticipate that the selectivity of these inhibitors will continue to enable the structural and mechanistic analyses of β-glucanases from diverse sources and protein families.

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Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Cited by 7 publications

References 35 publications

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B ₁ 4

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B ₁ 4

Covalent inhibitors of glycosidases and their applications in biochemistry and biology

Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases

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Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Cited by 7 publications

References 35 publications

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B 1 4

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B 1 4

Covalent inhibitors of glycosidases and their applications in biochemistry and biology

Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases

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Product

Resources

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Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B ₁ 4

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B ₁ 4