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Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11
Author(s) -
Xu Jianyong,
Ren Feifei,
Huang ChunHsiang,
Zheng Yingying,
Zhen Jie,
Sun Hong,
Ko TzuPing,
He Miao,
Chen ChunChi,
Chan HsiuChien,
Guo ReyTing,
Song Hui,
Ma Yanhe
Publication year - 2014
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24498
Subject(s) - pullulanase , thermostability , chemistry , pullulan , maltotriose , glycosidic bond , pyrococcus furiosus , stereochemistry , carbohydrate binding module , glycoside hydrolase , enzyme , biochemistry , maltose , polysaccharide , archaea , gene
Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K m for pullulan of PulA are 750 U mg −1 and 16.4 μmol L −1 , respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.