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New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases.

Tu T, Meng K, Luo H, Turunen O, Zhang L, Cheng Y, Su X, Ma R, Shi P, Wang Y, Yang P, Yao B - PLoS ONE (2015)

Bottom Line: In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions.Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75-4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3-20,000 fold) and kcat/Km (~8-187,500 fold) values and significantly increased △(△G) values (5.92-33.47 kJ/mol).Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.

ABSTRACT
Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75-4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3-20,000 fold) and kcat/Km (~8-187,500 fold) values and significantly increased △(△G) values (5.92-33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.

No MeSH data available.


Related in: MedlinePlus

The consensus sequence logo of the loop T3 regions of 1,000 GH28 endo-PGs generated using the WEBLOGO.
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pone.0135413.g005: The consensus sequence logo of the loop T3 regions of 1,000 GH28 endo-PGs generated using the WEBLOGO.

Mentions: According to the statistics drawn from the carbohydrate-active enzyme (CAZy) database [35], GH28 consists of 2,547 members from both microorganisms (archaea, bacteria and microbial eukaryotes) and plants. The vast majority of GH28 enzymes appear to be PGs, and 205 of them have been biochemically characterized. Using the protein sequence of PG8fn as a query, the Blast search generated a group of 1,000 complete sequences encoding putative GH28 endo-PGs of fungi. Further analysis of this GH28 subset revealed that the amino acid residue occupying position 94 is highly conserved as Asn in the T3 loop region (Fig 5), which is among the most flexible regions in PG [36]. The results indicated that the conserved residue of T3 loop, Asn94, might play an important role in the catalytic process. It is widely acknowledged that mobile loops in close proximity to the active site demonstrate pronounced conformational flexibility and contribute to enzyme catalysis. For example, the conformational change of a highly conserved thumb-loop of the GH11 xylanase close to the active site is directly associated with the binding of the substrate and the release of the product [19–21, 37–41]. In other words, the importance of the amino acid side chain at position 94 might be comparable to that of Ile116 at the tip of the thumb-loop of GH11 xylanase from Thermobacillus xylanilyticus [19–21], and that of W99 in the β2α2 loop region of GH51 arabinofuranosidase from T. xylanilyticus [22]. This is the first report on the role of T3 loop in the catalytic efficiency of GH28 endo-PG. Therefore, considering the above analysis, the residue Asn94 is an important position to investigate the effect of mutations on the catalytic efficiency.


New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases.

Tu T, Meng K, Luo H, Turunen O, Zhang L, Cheng Y, Su X, Ma R, Shi P, Wang Y, Yang P, Yao B - PLoS ONE (2015)

The consensus sequence logo of the loop T3 regions of 1,000 GH28 endo-PGs generated using the WEBLOGO.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556634&req=5

pone.0135413.g005: The consensus sequence logo of the loop T3 regions of 1,000 GH28 endo-PGs generated using the WEBLOGO.
Mentions: According to the statistics drawn from the carbohydrate-active enzyme (CAZy) database [35], GH28 consists of 2,547 members from both microorganisms (archaea, bacteria and microbial eukaryotes) and plants. The vast majority of GH28 enzymes appear to be PGs, and 205 of them have been biochemically characterized. Using the protein sequence of PG8fn as a query, the Blast search generated a group of 1,000 complete sequences encoding putative GH28 endo-PGs of fungi. Further analysis of this GH28 subset revealed that the amino acid residue occupying position 94 is highly conserved as Asn in the T3 loop region (Fig 5), which is among the most flexible regions in PG [36]. The results indicated that the conserved residue of T3 loop, Asn94, might play an important role in the catalytic process. It is widely acknowledged that mobile loops in close proximity to the active site demonstrate pronounced conformational flexibility and contribute to enzyme catalysis. For example, the conformational change of a highly conserved thumb-loop of the GH11 xylanase close to the active site is directly associated with the binding of the substrate and the release of the product [19–21, 37–41]. In other words, the importance of the amino acid side chain at position 94 might be comparable to that of Ile116 at the tip of the thumb-loop of GH11 xylanase from Thermobacillus xylanilyticus [19–21], and that of W99 in the β2α2 loop region of GH51 arabinofuranosidase from T. xylanilyticus [22]. This is the first report on the role of T3 loop in the catalytic efficiency of GH28 endo-PG. Therefore, considering the above analysis, the residue Asn94 is an important position to investigate the effect of mutations on the catalytic efficiency.

Bottom Line: In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions.Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75-4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3-20,000 fold) and kcat/Km (~8-187,500 fold) values and significantly increased △(△G) values (5.92-33.47 kJ/mol).Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.

ABSTRACT
Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75-4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3-20,000 fold) and kcat/Km (~8-187,500 fold) values and significantly increased △(△G) values (5.92-33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.

No MeSH data available.


Related in: MedlinePlus