title: Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase from Bacillus sp. HJB17.
abstract: β-Propeller phytases (BPPs) are abundant in nature. Recently, dual-domain BPPs have been found in which the typical BPP domain is responsible for phytate hydrolysis. The dual-domain BPP (PhyH) from Bacillus sp. HJB17 was obtained with an incomplete N-terminal BPP domain (PhyH-DI; residues 41-318) and a typical BPP domain (PhyH-DII; residues 319-644) at the C-terminus. PhyH-DI was found to act synergistically (with a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. The structure of PhyH was therefore studied with the aim of explaining these functions. PhyH with the secreted signal peptide of the first 40 amino acids deleted (PhyHT) was cloned and expressed in Escherichia coli. Purified and active PhyHT protein was obtained by refolding from the precipitant. PhyHT was crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 9.5, 25%(w/v) polyethylene glycol 4000 using 1 mg ml(-1) protein solution at 289 K. A complete data set was collected from a crystal to 2.85 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space group P1211, with unit-cell parameters a = 46.82, b = 140.19, c = 81.94 Å, α = 90.00, β = 92.00, γ = 90.00°. The asymmetric unit was estimated to contain one molecule of PhyHT.
authors.affiliation: College of Science, Beijiing Forestry University, 35 Qinghuadong Road, Haidian District, Beijing 100083, People's Republic of China.
authors.forename: Fang
authors.initials: F
authors.lastname: Lu
authors.affiliation: College of Science, Beijiing Forestry University, 35 Qinghuadong Road, Haidian District, Beijing 100083, People's Republic of China.
authors.forename: Gangxin
authors.initials: G
authors.lastname: Guo
authors.affiliation: College of Science, Beijiing Forestry University, 35 Qinghuadong Road, Haidian District, Beijing 100083, People's Republic of China.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Li
authors.affiliation: College of Science, Beijiing Forestry University, 35 Qinghuadong Road, Haidian District, Beijing 100083, People's Republic of China.
authors.forename: Duo
authors.initials: D
authors.lastname: Feng
authors.affiliation: College of Science, Beijiing Forestry University, 35 Qinghuadong Road, Haidian District, Beijing 100083, People's Republic of China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.affiliation: College of Science, Beijiing Forestry University, 35 Qinghuadong Road, Haidian District, Beijing 100083, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D010832:6-Phytase / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology*; D003461:Crystallography; D018360:Crystallography, X-Ray; D011487:Protein Conformation
chemical_list: D010832:6-Phytase
keywords: Bacillus sp. HJB17; PhyHT; β-propeller phytases