Changing Enzymic Reaction Mechanisms by Mutagenesis: Conversion of a Retaining Glucosidase to an Inverting Enzyme

Wang, Qingping; Graham, RM; Trimbur, D.; Warren, R. A. J.; Withers, S. G.

doi:10.1021/ja00104a060

Cited by 171 publications

(153 citation statements)

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“…Interestingly, formate had a greater effect on rate acceleration, although it exhibits lower nucleophilicity. This was also observed with the nucleophile-less mutants of the ␤-glucosidase from Agrobacterium faecalis (27) and the 1,3-1,4-␤-glucanase from Geobacillus licheniformis (28). It is possible that the strong resemblance between formate and the missing carboxylate nucleophile allows better accommodation of formate in the created cavity.…”

Section: Fig 2 Brønsted Plots Of the Hydrolysis Of Aryl ␤-D-xylopyrmentioning

confidence: 62%

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Bravman

Belakhov

Solomon

et al. 2003

Journal of Biological Chemistry

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␤-D-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a ␤-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased ϳ10 6 -fold, and this activity was enhanced 10 3 -fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu 335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK a ). The mutant exhibited 10 3 -fold reduction in activity, and the Brønsted plot of log(k cat ) versus pK a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 10 3 -fold. The rates of the glycosylation step, as reflected by the specificity constant (k cat /K m ), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK a ) but decreased 10 2 -fold for those that require strong acid catalysis (high pK a ). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp 495 acting as the acid-base in XynB2.

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Section: Fig 2 Brønsted Plots Of the Hydrolysis Of Aryl ␤-D-xylopyrmentioning

confidence: 62%

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Bravman

Belakhov

Solomon

et al. 2003

Journal of Biological Chemistry

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“…In this study, contamination from external sources seems unlikely, because the gel-filtration column and all other equipment involved were thoroughly washed with 0.5 M NaOH before each purification, and the elution buffer from the column was constantly monitored for activity and gave no detectable residual activity. One of the ways to detect and eliminate contaminant activities in glycoside hydrolases includes the use of mechanism-based inactivators, such as 2-deoxy-2-fluoroglycosides, which covalently bind to the carboxylic nucleophilic residue of the wild type enzyme (41,42,44,45). Here, we used an alternative approach to detect the presence of contaminant wild type AbfA T-6 in the E294A mutant sample.…”

Section: Figmentioning

confidence: 99%

“…The anomeric configuration of the product should always be characterized, because azide can also rescue the activity of mutants lacking the acid-base catalytic residue, but this time yielding the glycosyl-azide product with retained anomeric configuration. The rate enhancements achieved by the addition of azide to nucleophileless mutants of retaining glycoside hydrolases can range from 10-to 10 7 -fold and in some cases can regain the wild type activity (16,44,45,50).…”

Section: Figmentioning

confidence: 99%

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

Journal of Biological Chemistry

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␣-L-Arabinofuranosidases cleave the L-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl ␣-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K m value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2 ,4 ,6 -trichlorophenyl ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as ␤-L-arabinofuranosyl-azide by 1 H NMR, 13 C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and ␤-xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...

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“…The other family 19 enzymes from Streptomyces species and plants are also inverters. In the inverting enzymes, a large separation (> 10 Å ) has been generally found between the two carboxylic residues essential for the catalysis [36]. In the Aeromonas enzyme therefore the catalytic residues seem to be largely separated as well, hence the fine structure of the catalytic cleft should be conserved in this enzyme.…”

Section: Discussionmentioning

confidence: 99%

A novel type of family 19 chitinase from Aeromonas sp. No.10S‐24

Ueda

Kojima

Yoshikawa

et al. 2003

European Journal of Biochemistry

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A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced a-anomer by hydrolyzing b-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc) 6 ] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc) 2 and (GlcNAc) 4 . The evidence from this work suggested that the subsite structure of the enzyme was ()2)()1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure ()3)()2)()1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.Keywords: Aeromonas; chitinase; hydrolytic mechanism; oligosaccharide; subsites.Next to cellulose, chitin, b-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc), is one of the most commonly occurring organic compounds on earth, and attracts special interest as a reusable material. Chitinases (EC 3.2.1.14) degrade chitin by hydrolyzing the glycosidic linkages, and are widely distributed in various organisms [1]; from the ecological viewpoint, chitinases play an important role in recycling chitin in nature. The enzymes sequenced thus far are divided into two families (family 18 and family 19) on the basis of their amino acid sequences [2]. The chitinases of the two families do not share sequence similarity, and possess completely different folds in their three-dimensional structures. From X-ray crystallographic studies of the chitinases [3][4][5][6][7][8], the family 18 enzymes were found to have an (a/b) 8 -barrel fold, while the family 19 enzymes are rich in a-helices and composed of two lobes. When the b-1,4-glycosidic linkage is hydrolyzed by the catalytic action of the family 18 chitinases, oligosaccharide fragments with b-anomer are produced (retaining mechanism) [9], while the a-anomer is produced by the family 19 enzymes (inverting mechanism) [10]. Family 18 chitinases are sensitive to allosamidin, but family 19 chitinases are insensitive [11]. The family 18 enzymes have been found widely in bacteria, fungi, viruses, animals, and plants (classes III and V). Most of the family 19 chitinases, however, have been isolated from plants, and are subdivided into different classes (classes I, II and IV) [12]. The class I and IV chitinases have a cysteine-rich domain at their N-terminus, which is involved in substrate binding. Class IV chitinases are smaller than class I chitinases due to deletions in both the cysteine-rich domain and the catalytic domain. Class II chitinases are homologous to those of class I...

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Changing Enzymic Reaction Mechanisms by Mutagenesis: Conversion of a Retaining Glucosidase to an Inverting Enzyme

Cited by 171 publications

References 0 publications

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

A novel type of family 19 chitinase from Aeromonas sp. No.10S‐24

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