title: Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11.
abstract: 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 Km 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.
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Jianyong
authors.initials: J
authors.lastname: Xu
authors.forename: Feifei
authors.initials: F
authors.lastname: Ren
authors.forename: Chun-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zheng
authors.forename: Jie
authors.initials: J
authors.lastname: Zhen
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.forename: Miao
authors.initials: M
authors.lastname: He
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Chen
authors.forename: Hsiu-Chien
authors.initials: HC
authors.lastname: Chan
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
authors.forename: Hui
authors.initials: H
authors.lastname: Song
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D058677:Anoxybacillus / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000493:pharmacokinetics* / Q000648:ultrastructure*; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D005936:Glucans; D009844:Oligosaccharides; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: carbohydrate-binding motif; complex structure; crystal structure; pullulanase; thermostable