title: The tandemly repeated domains of a β-propeller phytase act synergistically to increase catalytic efficiency.
abstract: β-Propeller phytases (BPPs) with tandemly repeated domains are abundant in nature. Previous studies have shown that the intact domain is responsible for phytate hydrolysis, but the function of the other domain is relatively unknown. In this study, a new dual-domain BPP (PhyH) from Bacillus sp. HJB17 was identified to contain 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. Purified recombinant PhyH and PhyH-DII required Ca(2+) for phytase activity, showed activity at low temperatures (0-35 °C) and pH 6.0-8.0, and remained active (at 37 °C) after incubation at 60 °C and pH 6.0-12.0. Compared with PhyH-DII, PhyH is catalytically more active against phytate (catalytic constant 27.72 versus 4.17 s(-1)), which indicates the importance of PhyH-DI in phytate degradation. PhyH-DI was found to hydrolyze phytate intermediate D-Ins(1,4,5,6) P(4), and to act synergistically (a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. Furthermore, fusion of PhyH-DI with PhyP or 168PhyA significantly enhanced their catalytic efficiencies. This is the first report to elucidate the substrate specificity of the incomplete domain and the functional relationship of tandemly repeated domains in BPPs. We conjecture that dual-domain BPPs have succeeded evolutionarily because they can increase the amount of available phosphate by interacting together. Additionally, fusing PhyH-DI to a single-domain phytase appears to be an efficient way to improve the activity of the latter.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Li
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Tiezheng
authors.initials: T
authors.lastname: Yuan
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhao
authors.forename: Kun
authors.initials: K
authors.lastname: Meng
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001110:Arctic Regions; D001407:Bacillus / Q000201:enzymology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D002118:Calcium / Q000378:metabolism; D002681:China; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012988:Soil Microbiology; D013379:Substrate Specificity; D020080:Tandem Repeat Sequences; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007295:Inositol Phosphates; D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C081651:inositol-1,4,5,6-tetrakisphosphate; D010833:Phytic Acid; D010832:6-Phytase; D002118:Calcium