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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

Illustration of the substrate pentagalacturonic acid docked to the wild type PG8fn catalytic pocket.The system was constructed using PyMOL. The protein surface is shown in transparent gray. The catalytic region forms a tunnel through which the substrate passes. Hydrogen bond is depicted as blue dashed lines. Asn94 is marked in green. Catalytic triads in the active center are marked in cyan. The key amino acids interacted with GalpA at –1/+1 subsites are marked in orange.
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pone.0135413.g002: Illustration of the substrate pentagalacturonic acid docked to the wild type PG8fn catalytic pocket.The system was constructed using PyMOL. The protein surface is shown in transparent gray. The catalytic region forms a tunnel through which the substrate passes. Hydrogen bond is depicted as blue dashed lines. Asn94 is marked in green. Catalytic triads in the active center are marked in cyan. The key amino acids interacted with GalpA at –1/+1 subsites are marked in orange.

Mentions: For elucidation of the functional role of the mobile T3 loop of PG8fn in the recognition of the substrate, molecular docking studies were performed. The structure of PG8fn was modified through energy minimization, and the quality validation indicated that the comparative model is sufficient for further analysis. The docking results are listed in S2 Table. The predicted binding affinity of all binding modes is in the range from −9.3 to −8.0 kcal/mol, and the substrate pentagalacturonic acid has a very flexible conformation as shown by the changing calculated RMSD values relative to the best mode. After subsequent equilibration stable positions for the substrate were established, the conformation of PG8fn-pentagalacturonic acid complex (Fig 2) was generated based on the conformation of galacturonate units co-crystallized with S. purpureum endo-PG I (1KCD) [13] and the modeled structure of the polygalacturonase-octagalacturonate complex [12]. The active site of PG8fn is located at the bottom of a shallow groove enclosed by the β-strand PB1 and T1 and T3 loops. The pentagalacturonic acid docked into the SBP of the wild type PG8fn showed an extended conformation, representing a highly stable enzyme-substrate complex to allow the formation of productive complex and formation of the glycosyl-enzyme intermediate. According to the putative structure of PG8fn, Asp155, Asp176 and Asp177 form the catalytic triads corresponding to Asp153, Asp173 and Asp174 in S. purpureum endo-PG I [13], respectively. Therefore, Asp176 is the putative acid/base and Asp177 and Asp155 activate the water molecule that functions as a nucleophile in the inverting mechanism. Asp177 and Asp155 are closely located at the bottom of the active site with a separation of less than 5.0 Å, similar to that of A. aculeatus PG [12]. The non-reducing end of the substrate is directed toward the N terminus of the enzyme according to Pagès et al. [34]. GalpA at subsite –1/+1 interacts with Asn94, Gln124, His152, Asn153, Arg231, Lys233, and Tyr266 by forming hydrogen bonds. The O34 hydroxyl group of pentagalacturonic acid at the subsite +1 forms a hydrogen bond with the ND2 of Asn94 (2.5 Å), which is consistent with the crystal structures of S. purpureum endo-PG I complex [13]. These results highlight the important role of Asn94 of T3 loop in substrate binding.


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)

Illustration of the substrate pentagalacturonic acid docked to the wild type PG8fn catalytic pocket.The system was constructed using PyMOL. The protein surface is shown in transparent gray. The catalytic region forms a tunnel through which the substrate passes. Hydrogen bond is depicted as blue dashed lines. Asn94 is marked in green. Catalytic triads in the active center are marked in cyan. The key amino acids interacted with GalpA at –1/+1 subsites are marked in orange.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135413.g002: Illustration of the substrate pentagalacturonic acid docked to the wild type PG8fn catalytic pocket.The system was constructed using PyMOL. The protein surface is shown in transparent gray. The catalytic region forms a tunnel through which the substrate passes. Hydrogen bond is depicted as blue dashed lines. Asn94 is marked in green. Catalytic triads in the active center are marked in cyan. The key amino acids interacted with GalpA at –1/+1 subsites are marked in orange.
Mentions: For elucidation of the functional role of the mobile T3 loop of PG8fn in the recognition of the substrate, molecular docking studies were performed. The structure of PG8fn was modified through energy minimization, and the quality validation indicated that the comparative model is sufficient for further analysis. The docking results are listed in S2 Table. The predicted binding affinity of all binding modes is in the range from −9.3 to −8.0 kcal/mol, and the substrate pentagalacturonic acid has a very flexible conformation as shown by the changing calculated RMSD values relative to the best mode. After subsequent equilibration stable positions for the substrate were established, the conformation of PG8fn-pentagalacturonic acid complex (Fig 2) was generated based on the conformation of galacturonate units co-crystallized with S. purpureum endo-PG I (1KCD) [13] and the modeled structure of the polygalacturonase-octagalacturonate complex [12]. The active site of PG8fn is located at the bottom of a shallow groove enclosed by the β-strand PB1 and T1 and T3 loops. The pentagalacturonic acid docked into the SBP of the wild type PG8fn showed an extended conformation, representing a highly stable enzyme-substrate complex to allow the formation of productive complex and formation of the glycosyl-enzyme intermediate. According to the putative structure of PG8fn, Asp155, Asp176 and Asp177 form the catalytic triads corresponding to Asp153, Asp173 and Asp174 in S. purpureum endo-PG I [13], respectively. Therefore, Asp176 is the putative acid/base and Asp177 and Asp155 activate the water molecule that functions as a nucleophile in the inverting mechanism. Asp177 and Asp155 are closely located at the bottom of the active site with a separation of less than 5.0 Å, similar to that of A. aculeatus PG [12]. The non-reducing end of the substrate is directed toward the N terminus of the enzyme according to Pagès et al. [34]. GalpA at subsite –1/+1 interacts with Asn94, Gln124, His152, Asn153, Arg231, Lys233, and Tyr266 by forming hydrogen bonds. The O34 hydroxyl group of pentagalacturonic acid at the subsite +1 forms a hydrogen bond with the ND2 of Asn94 (2.5 Å), which is consistent with the crystal structures of S. purpureum endo-PG I complex [13]. These results highlight the important role of Asn94 of T3 loop in substrate binding.

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