The identification of the acid–base catalyst of α‐arabinofuranosidase from <i>Geobacillus stearothermophilus</i> T‐6, a family 51 glycoside hydrolase

Shallom, D.; Belakhov, V. V.; Solomon, Dmitry; Gilead-Gropper, Sara; Baasov, Timor; Shoham, G.; Shoham, Yuval

doi:10.1016/s0014-5793(02)02343-8

Cited by 79 publications

(74 citation statements)

References 31 publications

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“…In a previous work done on AbfA T-6, we took advantage of the observation that the enzyme has low, yet sufficient, activity on aryl-␤-D-xylopyranosides to perform preliminary kinetic analysis of the wild type and E175A mutant with several such substrates (19). For this study we prepared several aryl-␣-L-arabinofuranosides with pK a values of the phenol leaving group ranging from 5.15 to 8.39.…”

Section: Resultsmentioning

confidence: 99%

“…Cloning, Mutagenesis, Protein Expression, and Purification-The abfA T-6 gene (GenBank TM accession number AF159625) from G. stearothermophilus T-6 was cloned in the pET9d vector and overexpressed in Escherichia coli BL21(DE3) as previously reported (19). Site-directed mutagenesis was performed using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The mutated genes were sequenced to confirm that only the desired mutations were inserted. The wild type and mutated proteins were overexpressed and purified as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

“…Both catalytic mechanisms are found in the different ␣-L-arabinofuranosidases families: GH51 and GH54 were shown to cleave the glycosidic bond with a retention of the anomeric configuration (15); GH43, which includes ␣-L-arabinofuranosidases as well as ␤-D-xylosidases, was shown to work via the inverting mechanism (15), whereas the stereochemistry of GH62 is not yet characterized. Currently there are 32 sequences of ␣-L-arabinofuranosidases classified as GH51 glycoside hydrolases, 2 and, although much work was done on the substrate specificities of these enzymes, there are only a few studies concerning the biochemical mechanism and catalytic properties of this family (17)(18)(19). Based on sequence similarities, GH51 is part of the GH-A superfamily (20), suggesting that the active site of the enzymes of this family is located at the C-terminal end of a (␤/␣) 8 fold.…”

mentioning

confidence: 99%

“…The ␣-L-arabinofuranosidases display several unique features making them attractive candidates for glycosynthesis. These enzymes belong to the few glycoside hydrolase families that are able to hydrolyze a furanosidic substrate; several ␣-L-arabinofuranosidases can also hydrolyze aryl ␤-Dxylopyranosides, thus their glycosynthase mutants would probably be able to accept both arabinofuranosidases and xylopyranosides as donors (6,19). To the best of our knowledge, such engineered furanosidic-glycosynthases have not been reported yet.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The mutated genes were sequenced to confirm that only the desired mutations were inserted. The wild type and mutated proteins were overexpressed and purified as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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Improved activity of α‐L‐arabinofuranosidase from Geobacillus vulcani GS90 by directed evolution: Investigation on thermal and alkaline stability

Sürmeli

İlgü

Şanlı‐Mohamed

2018

Biotech and App Biochem

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α-L-Arabinofuranosidase (Abf) is a potential enzyme because of its synergistic effect with other hemicellulases in agro-industrial field. In this study, directed evolution was applied to Abf from Geobacillus vulcani GS90 (GvAbf) using one round error-prone PCR and constructed a library of 73 enzyme variants of GvAbf. The activity screening of the enzyme variants was performed on soluble protein extracts using p-nitrophenyl α-L-arabinofuranoside as substrate. Two high activity displaying variants (GvAbf L307S and GvAbf Q90H/L307S) were selected, purified, partially characterized, and structurally analyzed. The specific activities of both variants were almost 2.5-fold more than that of GvAbf. Both GvAbf variants also exhibited higher thermal stability but lower alkaline stability in reference to GvAbf. The structural analysis of GvAbf model indicated that two mutation sites Q90H and L307S in both GvAbf variants are located in TIM barrel domain, responsible for catalytic action in many Glycoside Hydrolase Families including GH51. The structure of GvAbf model displayed that the position of L307S mutation is closer to the catalytic residues of GvAbf compared with Q90H mutation and also L307S mutation is conserved in both variants of GvAbf. Therefore, it was hypothesized that L307S amino acid substitution may play a critical role in catalytic activity of GvAbf.

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Structural and functional analyses of GH51 alpha‐L‐arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability

Sürmeli

Şanlı‐Mohamed

2022

Biotech and App Biochem

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Alpha‐L‐arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most‐active and the least‐active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid–base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C–C bond between C171 and C213 by becoming closer to each other.

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The identification of the acid–base catalyst of α‐arabinofuranosidase from Geobacillus stearothermophilus T‐6, a family 51 glycoside hydrolase

Cited by 79 publications

References 31 publications

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

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

Improved activity of α‐L‐arabinofuranosidase from Geobacillus vulcani GS90 by directed evolution: Investigation on thermal and alkaline stability

Structural and functional analyses of GH51 alpha‐L‐arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability

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