Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis
Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosy...
Ausführliche Beschreibung
Autor*in: |
Khan, Samiullah [verfasserIn] |
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Englisch |
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2011 |
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Anmerkung: |
© Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
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Übergeordnetes Werk: |
Enthalten in: BMC biochemistry - London : BioMed Central, 2000, 12(2011), 1 vom: 23. Feb. |
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Übergeordnetes Werk: |
volume:12 ; year:2011 ; number:1 ; day:23 ; month:02 |
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DOI / URN: |
10.1186/1471-2091-12-11 |
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Katalog-ID: |
SPR02681692X |
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100 | 1 | |a Khan, Samiullah |e verfasserin |4 aut | |
245 | 1 | 0 | |a Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
264 | 1 | |c 2011 | |
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500 | |a © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( | ||
520 | |a Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. | ||
650 | 4 | |a Quercetin |7 (dpeaa)DE-He213 | |
650 | 4 | |a Triple Mutant |7 (dpeaa)DE-He213 | |
650 | 4 | |a Cellotetraose |7 (dpeaa)DE-He213 | |
650 | 4 | |a Catalytic Cleft |7 (dpeaa)DE-He213 | |
650 | 4 | |a Quercetin Glucoside |7 (dpeaa)DE-He213 | |
700 | 1 | |a Pozzo, Tania |4 aut | |
700 | 1 | |a Megyeri, Márton |4 aut | |
700 | 1 | |a Lindahl, Sofia |4 aut | |
700 | 1 | |a Sundin, Anders |4 aut | |
700 | 1 | |a Turner, Charlotta |4 aut | |
700 | 1 | |a Karlsson, Eva Nordberg |4 aut | |
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912 | |a GBV_ILN_2027 | ||
912 | |a GBV_ILN_4012 | ||
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10.1186/1471-2091-12-11 doi (DE-627)SPR02681692X (SPR)1471-2091-12-11-e DE-627 ger DE-627 rakwb eng Khan, Samiullah verfasserin aut Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. Quercetin (dpeaa)DE-He213 Triple Mutant (dpeaa)DE-He213 Cellotetraose (dpeaa)DE-He213 Catalytic Cleft (dpeaa)DE-He213 Quercetin Glucoside (dpeaa)DE-He213 Pozzo, Tania aut Megyeri, Márton aut Lindahl, Sofia aut Sundin, Anders aut Turner, Charlotta aut Karlsson, Eva Nordberg aut Enthalten in BMC biochemistry London : BioMed Central, 2000 12(2011), 1 vom: 23. Feb. (DE-627)326179399 (DE-600)2041216-2 1471-2091 nnns volume:12 year:2011 number:1 day:23 month:02 https://dx.doi.org/10.1186/1471-2091-12-11 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2011 1 23 02 |
spelling |
10.1186/1471-2091-12-11 doi (DE-627)SPR02681692X (SPR)1471-2091-12-11-e DE-627 ger DE-627 rakwb eng Khan, Samiullah verfasserin aut Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. Quercetin (dpeaa)DE-He213 Triple Mutant (dpeaa)DE-He213 Cellotetraose (dpeaa)DE-He213 Catalytic Cleft (dpeaa)DE-He213 Quercetin Glucoside (dpeaa)DE-He213 Pozzo, Tania aut Megyeri, Márton aut Lindahl, Sofia aut Sundin, Anders aut Turner, Charlotta aut Karlsson, Eva Nordberg aut Enthalten in BMC biochemistry London : BioMed Central, 2000 12(2011), 1 vom: 23. Feb. (DE-627)326179399 (DE-600)2041216-2 1471-2091 nnns volume:12 year:2011 number:1 day:23 month:02 https://dx.doi.org/10.1186/1471-2091-12-11 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2011 1 23 02 |
allfields_unstemmed |
10.1186/1471-2091-12-11 doi (DE-627)SPR02681692X (SPR)1471-2091-12-11-e DE-627 ger DE-627 rakwb eng Khan, Samiullah verfasserin aut Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. Quercetin (dpeaa)DE-He213 Triple Mutant (dpeaa)DE-He213 Cellotetraose (dpeaa)DE-He213 Catalytic Cleft (dpeaa)DE-He213 Quercetin Glucoside (dpeaa)DE-He213 Pozzo, Tania aut Megyeri, Márton aut Lindahl, Sofia aut Sundin, Anders aut Turner, Charlotta aut Karlsson, Eva Nordberg aut Enthalten in BMC biochemistry London : BioMed Central, 2000 12(2011), 1 vom: 23. Feb. (DE-627)326179399 (DE-600)2041216-2 1471-2091 nnns volume:12 year:2011 number:1 day:23 month:02 https://dx.doi.org/10.1186/1471-2091-12-11 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2011 1 23 02 |
allfieldsGer |
10.1186/1471-2091-12-11 doi (DE-627)SPR02681692X (SPR)1471-2091-12-11-e DE-627 ger DE-627 rakwb eng Khan, Samiullah verfasserin aut Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. Quercetin (dpeaa)DE-He213 Triple Mutant (dpeaa)DE-He213 Cellotetraose (dpeaa)DE-He213 Catalytic Cleft (dpeaa)DE-He213 Quercetin Glucoside (dpeaa)DE-He213 Pozzo, Tania aut Megyeri, Márton aut Lindahl, Sofia aut Sundin, Anders aut Turner, Charlotta aut Karlsson, Eva Nordberg aut Enthalten in BMC biochemistry London : BioMed Central, 2000 12(2011), 1 vom: 23. Feb. (DE-627)326179399 (DE-600)2041216-2 1471-2091 nnns volume:12 year:2011 number:1 day:23 month:02 https://dx.doi.org/10.1186/1471-2091-12-11 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2011 1 23 02 |
allfieldsSound |
10.1186/1471-2091-12-11 doi (DE-627)SPR02681692X (SPR)1471-2091-12-11-e DE-627 ger DE-627 rakwb eng Khan, Samiullah verfasserin aut Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. Quercetin (dpeaa)DE-He213 Triple Mutant (dpeaa)DE-He213 Cellotetraose (dpeaa)DE-He213 Catalytic Cleft (dpeaa)DE-He213 Quercetin Glucoside (dpeaa)DE-He213 Pozzo, Tania aut Megyeri, Márton aut Lindahl, Sofia aut Sundin, Anders aut Turner, Charlotta aut Karlsson, Eva Nordberg aut Enthalten in BMC biochemistry London : BioMed Central, 2000 12(2011), 1 vom: 23. Feb. (DE-627)326179399 (DE-600)2041216-2 1471-2091 nnns volume:12 year:2011 number:1 day:23 month:02 https://dx.doi.org/10.1186/1471-2091-12-11 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2011 1 23 02 |
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Enthalten in BMC biochemistry 12(2011), 1 vom: 23. Feb. volume:12 year:2011 number:1 day:23 month:02 |
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Khan, Samiullah @@aut@@ Pozzo, Tania @@aut@@ Megyeri, Márton @@aut@@ Lindahl, Sofia @@aut@@ Sundin, Anders @@aut@@ Turner, Charlotta @@aut@@ Karlsson, Eva Nordberg @@aut@@ |
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. 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Khan, Samiullah |
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Khan, Samiullah misc Quercetin misc Triple Mutant misc Cellotetraose misc Catalytic Cleft misc Quercetin Glucoside Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
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Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis Quercetin (dpeaa)DE-He213 Triple Mutant (dpeaa)DE-He213 Cellotetraose (dpeaa)DE-He213 Catalytic Cleft (dpeaa)DE-He213 Quercetin Glucoside (dpeaa)DE-He213 |
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Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
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Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
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aglycone specificity of thermotoga neapolitana β-glucosidase 1a modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
title_auth |
Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
abstract |
Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
abstractGer |
Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
abstract_unstemmed |
Background The thermostable β-glucosidase (Tn Bgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of Tn Bgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (p NPGlc), and screened for hydrolysis of Q3. Results Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as $ T_{m} $ by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5 - 10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in $ K_{M} $ and turnover. The $ K_{M} $-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for p NPGlc, while the $ K_{M} $ decreased a corresponding extent for Q3. Turnover was only significantly changed using p NPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in $ K_{M} $ for this substrate. Conclusion These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both $ K_{M} $ and turnover. An affinity change, leading to a decreased $ K_{M} $, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site. © Khan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
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Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis |
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