Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase fromBacillussp. HJB17
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) fromBacillussp. 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 inEscherichia 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 Msodium acetate trihydrate, 0.1 MTris–HCl pH 9.5, 25%(w/v) polyethylene glycol 4000 using 1 mg ml−1protein 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 groupP1211, with unit-cell parametersa= 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.