Stereochemistry of family 52 glycosyl hydrolases: A β-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme

Tsafrir Bravman; Gennady Zolotnitsky; Smadar Shulami; Valery Belakhov; Dmitry Solomon; Timor Baasov; Gil Shoham; Yuval Shoham

doi:10.1016/S0014-5793(01)02360-2

Stereochemistry of family 52 glycosyl hydrolases: A β-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme

Tsafrir Bravman, Gennady Zolotnitsky, Smadar Shulami, Valery Belakhov, Dmitry Solomon, Timor Baasov, Gil Shoham, Yuval Shoham^*

^*Corresponding author for this work

Institute of Chemistry

Research output: Contribution to journal › Article › peer-review

42 Scopus citations

Abstract

A β-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65°C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl β-D-xylopyranoside was followed by ¹H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

Original language	English
Pages (from-to)	39-43
Number of pages	5
Journal	FEBS Letters
Volume	495
Issue number	1-2
DOIs	https://doi.org/10.1016/S0014-5793(01)02360-2
State	Published - 20 Apr 2001

Bibliographical note

Funding Information:
This study was supported by Grants from the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem) (no. 676/00 to G.S. and Y.S.), and from the United States–Israel Binational Science Foundation (BSF) (no. 96-178 to Y.S.), Jerusalem, Israel. Additional support was provided by the Fund for the Promotion of Research at the Technion, and by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany). V.B. acknowledges the financial support by the Center of Absorption in Science, the Ministry of Immigration Absorption and the Ministry of Science and Arts, Israel (Kamea Program).

Keywords

Bacillus stearothermophilus
Catalytic residue
Glycosyl hydrolase family 52
Nuclear magnetic resonance
Retaining mechanism
β-Xylosidase

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10.1016/S0014-5793(01)02360-2

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title = "Stereochemistry of family 52 glycosyl hydrolases: A β-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme",

abstract = "A β-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65°C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl β-D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.",

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author = "Tsafrir Bravman and Gennady Zolotnitsky and Smadar Shulami and Valery Belakhov and Dmitry Solomon and Timor Baasov and Gil Shoham and Yuval Shoham",

note = "Funding Information: This study was supported by Grants from the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem) (no. 676/00 to G.S. and Y.S.), and from the United States–Israel Binational Science Foundation (BSF) (no. 96-178 to Y.S.), Jerusalem, Israel. Additional support was provided by the Fund for the Promotion of Research at the Technion, and by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany). V.B. acknowledges the financial support by the Center of Absorption in Science, the Ministry of Immigration Absorption and the Ministry of Science and Arts, Israel (Kamea Program).",

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AU - Zolotnitsky, Gennady

AU - Shulami, Smadar

AU - Belakhov, Valery

AU - Solomon, Dmitry

AU - Baasov, Timor

AU - Shoham, Gil

AU - Shoham, Yuval

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N2 - A β-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65°C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl β-D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

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Stereochemistry of family 52 glycosyl hydrolases: A β-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme

Abstract

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Keywords

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