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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

Root mean square deviation (RMSD) analysis of the wild type PG8fn (black), N94Q (red) and N94A (green) during a 50-ns MD simulation.(A) The enzyme backbones. (B) The pentagalacturonic acid. Each simulation was repeated three times with the same initial configurations.
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pone.0135413.g003: Root mean square deviation (RMSD) analysis of the wild type PG8fn (black), N94Q (red) and N94A (green) during a 50-ns MD simulation.(A) The enzyme backbones. (B) The pentagalacturonic acid. Each simulation was repeated three times with the same initial configurations.

Mentions: The RMSD values of the protein backbone atoms against the starting structures during the full MD simulation showed that all complex systems became dynamically equilibrated after 30 ns of simulation (Fig 3A). Both substitutions showed increased conformational flexibility with higher RMSD values at the temperature of 300 K. The two abrupt changes in RMSD values of the two mutant complexes are related to the equilibration protocol, in which the entire protein backbone had its position constrained initially, but the atoms of the protein backbone close to the substrate remained constrained in the second step only. The ligand RMSD depicted in Fig 3B showed that the pentagalacturonic acid in both mutant complexes is much more flexible in the catalytic site than that in the wild type. It’s the substrate detachment and rotation to account for the dissociation of pentagalacturonic acid from the protein.


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)

Root mean square deviation (RMSD) analysis of the wild type PG8fn (black), N94Q (red) and N94A (green) during a 50-ns MD simulation.(A) The enzyme backbones. (B) The pentagalacturonic acid. Each simulation was repeated three times with the same initial configurations.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135413.g003: Root mean square deviation (RMSD) analysis of the wild type PG8fn (black), N94Q (red) and N94A (green) during a 50-ns MD simulation.(A) The enzyme backbones. (B) The pentagalacturonic acid. Each simulation was repeated three times with the same initial configurations.
Mentions: The RMSD values of the protein backbone atoms against the starting structures during the full MD simulation showed that all complex systems became dynamically equilibrated after 30 ns of simulation (Fig 3A). Both substitutions showed increased conformational flexibility with higher RMSD values at the temperature of 300 K. The two abrupt changes in RMSD values of the two mutant complexes are related to the equilibration protocol, in which the entire protein backbone had its position constrained initially, but the atoms of the protein backbone close to the substrate remained constrained in the second step only. The ligand RMSD depicted in Fig 3B showed that the pentagalacturonic acid in both mutant complexes is much more flexible in the catalytic site than that in the wild type. It’s the substrate detachment and rotation to account for the dissociation of pentagalacturonic acid from the protein.

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