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

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dc.contributor Aalto-yliopisto fi
dc.contributor Aalto University en Tu, Tao Meng, Kun Luo, Huiying Turunen, Ossi Zhang, Lujia Cheng, Yanli Su, Xiaoyun Ma, Rui Shi, Pengjun Wang, Yaru Yang, Peilong Yao, Bin 2017-05-11T09:08:25Z 2017-05-11T09:08:25Z 2015
dc.identifier.citation Tu , T , Meng , K , Luo , H , Turunen , O , Zhang , L , Cheng , Y , Su , X , Ma , R , Shi , P , Wang , Y , Yang , P & Yao , B 2015 , ' New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases ' PLoS One , vol 10 , no. 9 , e0135413 , pp. 1-16 . DOI: 10.1371/journal.pone.0135413 en
dc.identifier.issn 1932-6203
dc.identifier.other PURE UUID: d2935228-f77c-459f-8fb4-cc5fae182159
dc.identifier.other PURE ITEMURL:
dc.identifier.other PURE FILEURL:
dc.description.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 thelonger 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. en
dc.format.extent 1-16
dc.format.mimetype application/pdf
dc.language.iso en en
dc.relation.ispartofseries PLoS One en
dc.relation.ispartofseries Volume 10, issue 9 en
dc.rights openAccess en
dc.subject.other 215 Chemical engineering en
dc.subject.other 220 Industrial biotechnology en
dc.subject.other 216 Materials engineering en
dc.title New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases en
dc.type A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä fi
dc.description.version Peer reviewed en
dc.contributor.department Department of Biotechnology and Chemical Technology en
dc.contributor.department Department of Bioproducts and Biosystems en
dc.subject.keyword 215 Chemical engineering
dc.subject.keyword 220 Industrial biotechnology
dc.subject.keyword 216 Materials engineering
dc.identifier.urn URN:NBN:fi:aalto-201705114251
dc.identifier.doi 10.1371/journal.pone.0135413
dc.type.version publishedVersion

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