The tandemly repeated domains of a b-propeller phytase act synergistically to increase catalytic efficiency Zhongyuan Li*, Huoqing Huang*, Peilong Yang, Tiezheng Yuan, Pengjun Shi, Junqi Zhao, Kun Meng and Bin Yao Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China Keywords dual domain; fusion protein; phytate; synergistic catalysis; b-propeller phytase Correspondence B. Yao, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, China Fax: +86 10 8210 6054 Tel: +86 10 8210 6053 E-mail: yaobin@caas-bio.net.cn; yaobin@mail.caas.net.cn *Z. Li and H. Huang contributed equally to this paper (Received 10 March 2011, revised 20 June 2011, accepted 23 June 2011) doi:10.1111/j.1742-4658.2011.08223.x b-Propeller phytases (BPPs) with tandemly repeated domains are abundant in nature. Previous studies have shown that the intact domain is responsi- ble for phytate hydrolysis, but the function of the other domain is rela- tively 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 tem- peratures (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 degrada- tion. 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 sig- nificantly enhanced their catalytic efficiencies. This is the first report to elu- cidate 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. Introduction Phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate, InsP 6 ) is the most abundant organic phosphorus compound in nature [1,2]. Microbial mineralization of phytate by phytase plays a significant role in the process of phosphorus recycling. InsP 6 can be hydrolyzed com- pletely to produce one inositol and six molecules of inorganic phosphate, or partially to produce lower inositol polyphosphate (IPP) isomers and inorganic phosphates [3]. Among the four types of phytases that have been identified, b-propeller phytase (BPP, EC 3.1.3.8 or EC 3.1.3.26) differs from the other three phytases (his- tidine acid phosphatase (HAP), cysteine phytase and purple acid phosphatase) by having a neutral (pH 7.0) rather than acidic pH optimum. Previous studies have shown that BPP is the major class of phytate- degrading enzyme in nature, which is widespread in terrestrial and aquatic ecosystems [4,5]. Until now, Abbreviations BPP, b-propeller phytase; HAP, histidine acid phosphatase; InsP 6 , myo-inositol hexakisphosphate; IPP, inositol polyphosphate; IPTG, isopropyl-b- D-1-thiogalactopyranoside. 3032 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS only a small number of BPPs have been isolated and studied, including Shewanella oneidensis MR-1 PhyS [6], Bacillus subtilis PhyC [7], Bacillus sp. DS11 Phy [8], B. subtilis 168 168PhyA [9], Bacillus licheniformis PhyL [9], Pedobacter nyackensis MJ11 PhyP [10] and Janthinobacterium sp. TN115 PhyA115 [11], all of which are mesophilic or thermophilic (37–70 °C). A typical BPP has a six-bladed propeller fold with two phosphate-binding sites (cleavage site and affinity site) and six calcium-binding sites, three of which are high-affinity binding sites responsible for enzyme sta- bility and three are low-affinity sites regulating the cat- alytic activity of the enzyme [12,13]. BPP prefers the hydrolysis of every second phosphate over adjacent ones, and degrades InsP 6 gradually into InsP 5 , InsP 4 , and the final product – Ins(2,4,6)P 3 and Ins(1,3,5)P 3 – via two alternative pathways [14]. Bacterial BPPs containing two tandemly repeated domains (dual domains) within a continuous sequence have been found in S. oneidensis MR-1 PhyS [6] and Janthinobacterium sp. TN115 [11]. However, few stud- ies have been reported concerning the functions and relationship of the dual domains. Here we describe the physical properties that relate to the catalysis of a newly isolated, dual-domain BPP (PhyH) from Bacillus sp. HJB17. This enzyme is very active at neutral pH (6.0–8.0) and at low temperatures (0–35 °C). We focus on the function and relationship of the two single domains. Additionally, the possibility that fusion of the PhyH N-terminal domain to other single-domain BPPs would improve the catalytic efficiency was assessed. Results Microorganism isolation Using phytase screening and low phosphate media, three strains with phytase activity were isolated from the alpine tundra soil of China No. 1 Glacier in Xinji- ang, China. Strain HJB17 exhibited the greatest phy- tase activity, 0.33 ± 0.05 UÆmL )1 , under optimal growth conditions (pH 7.0 and 37 °C). According to its 16S rDNA gene sequence (HQ610835), strain HJB17 belongs to the genus Bacillus (99% 16S rDNA gene sequence identity with that of Bacillus sp. SW41, HM584798.1), and has been deposited in the Agricul- tural Culture Collection of China under registration number ACCC 05550. Cloning and sequencing of BPP gene phyH phyH (HM003046) was amplified using degenerate PCR and thermal asymmetric interlaced PCR tech- niques. The full-length gene contains 1932 base pairs (643 amino acids). The deduced amino acid sequence (PhyH) contains a putative signal peptide (40 amino acids), an N-terminal domain (PhyH-DI, residues 41– 318) which shares 25% identity with that of Bacil- lus amyloliquefaciens TS-Phy (YP090097), and a C-ter- minal domain (PhyH-DII, residues 319–644) that has 49% identity with that of TS-Phy (YP090097) [15] and 41% with B. subtilis PhyC (CAM58513) [7]. Sequence alignment of PhyH-DI and PhyH-DII with five homo- logs identified six conserved residues (Pro30, Gly153, Gln157, Asp188, Ala208 and Gly234). Some of the res- idues involved in phosphate and calcium binding are conserved in the PhyH-DII sequence, whereas only one phosphate-binding residue is conserved in the PhyH-DI sequence (Fig. S1). There are two pairs of cysteine residues, Cys142 and Cys193 in PhyH-DII and Cys445 and Cys495 in PhyH-DI. These residues are at the same positions as Cys157, Cys206, Cys450 and Cys498 of PhyS [6], which form homologous disul- fide bonds to stabilize BPPs [16]. The three-dimensional structures of PhyH-DI and PhyH-DII were modeled using TS-Phy [15] as the tem- plate (data not shown). PhyH-DI was predicted to have a five-blade propeller structure and PhyH-DII a six-blade b-propeller containing five four-stranded sheets and one five-stranded sheet. Expression and purification of PhyH, PhyH-DI and PhyH-DII PhyH, PhyH-DI and PhyH-DII were each expressed in Escherichia coli. After isopropyl-b-d-1-thiogalactopyr- anoside (IPTG) induction at 20 °C for 20 h, substan- tial phytase activity was detected in all cultures, and no activity was detected in cultures that harbored an empty pET-22b(+). PhyH, PhyH-DI and PhyH-DII were purified to homogeneity by Ni-affinity chroma- tography and were found to have apparent molecular weights of 67.0, 31.0 and 36.0 kDa (Fig. 1A), respec- tively. Native gradient gel electrophoresis (Fig. 1B) demonstrated that native PhyH might be a dimer. The specific activities of PhyH and PhyH-DII against InsP 6 were 4.43 ± 0.55 and 1.82 ± 0.23 UÆmg )1 , respec- tively, at 35 °C. At the same temperature, PhyH-DI had no activity against InsP 6 . Biochemical properties of PhyH and PhyH-DII Ca 2+ is required for BPP activity, and the optimal concentration of Ca 2+ for the phytase activities of PhyH and PhyH-DII was 1 mm (Fig. 2A). Both enzymes exhibited optimal activities at pH 7.0, and Z. Li et al. Catalysis of a dual-domain b-propeller phytase FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3033 their apparent optimal temperatures were found to be 35 °C (Fig. 2B,C). At 0 °C, PhyH and PhyH-DII retained 22% and 15.6% of their maximum activities, respectively. PhyH appears to be the first BPP found to be active at such a low temperature. PhyH was sta- ble at neutral and alkaline pH; PhyH-DII was less sta- ble under the same conditions (Fig. 2D). PhyH was basically stable at 35 °C, and retained 60% of the initial activity at 45 °C for 90 min when assayed at 35 °C (Fig. 3A,B). The presence of Ca 2+ increased the thermal stability of PhyH and PhyH-DII. After incubation at 60 °C for 30 min, both enzymes retained > 70% of their initial activity in the presence of 10 mm Ca 2+ and < 30% of their activity without Ca 2+ (Fig. 3C,D). PhyH at 35 °C showed a spectrum (Fig. 4) with a weak minimum around 255 nm and a large positive maximum around 298 nm. The structure was intact after 12 h of incubation and failed to refold after 5 min of boiling. The kinetic values for PhyH and PhyH-DII towards InsP 6 at 35 °C and 20 °C are given in Table 1. The higher specific activity k cat and V max values and lower K m value of PhyH suggests that PhyH is more catalytically efficient and has a greater affinity for InsP 6 than PhyH-DII. Substrate specificity of PhyH-DI To understand the function of PhyH-DI, the substrate specificities of PhyH-DI against several IPPs including d-Ins(2)P 1 , d-Ins(1,4)P 2 , d-Ins(1,4,5)P 3 , d-Ins(1,4,5,6)P 4 and Ins(1,3,4,5,6)P 5 were determined. PhyH-DI has a specific activity of 4.28 ± 0.56 UÆmg )1 against d-Ins(1,4,5,6)P 4 at 35 °C and cannot hydrolyze other IPPs. The function of PhyH-DI in InsP 6 degradation When PhyH-DI was added into the reaction system of PhyH-DII and InsP 6 , 1.63 fold phosphate was released over that of PhyH-DII alone. More phosphate (1.17– 2.49 fold) was also released after subsequent addition of PhyH-DI to a BPP (168PhyA or PhyP), or an HAP (E. coli AppA) and InsP 6 reaction mixture (Table 2), Fig. 1. Electrophoretic analysis of PhyH, PhyH-DI and PhyH-DII. (A) SDS ⁄ PAGE analysis of purified PhyH, PhyH-DI and PhyH-DII. Lane M, molecular weight markers; lane 1, culture supernatant of PhyH; lane 2, culture supernatant of PhyH-DII; lane 3, culture supernatant of PhyH-DI; lane 4, purified PhyH; lane 5, purified PhyH-DII; and lane 6, purified PhyH-DI. (B) Non-denaturing gradient PAGE of PhyH. Lane M, native high molecular weight markers; lane 1, native PhyH stained with Coomassie Brilliant Blue R250. 0 20 40 60 80 100 120 3456789101112 pH of incubation Relative activity (%) PhyH PhyH-DII 0 20 40 60 80 100 120 012345 Concentration of Ca 2+ (m M ) Relative activity (%) PhyH PhyH-DII 0 20 40 60 80 100 120 345678910 pH Relative activity (%) PhyH PhyH-DII 0 20 40 60 80 100 120 0 102030405060 Temperature ( 嘙 C ) Relative activity (%) PhyH PhyH-DII AB CD Fig. 2. Environmental factors that affect the catalytic activities of PhyH and PhyH-DII. (A) Effect of 0–5.0 mM Ca 2+ at 37 °C. (B) Effect of pH at 37 °C. (C) Effect of temperature at pH 7.0. (D) pH stability. Residual activity was assayed under optimal conditions (1 m M Ca 2+ ,35°C, pH 7.0) after incubation in buffers at 37 °C for 1 h. Catalysis of a dual-domain b-propeller phytase Z. Li et al. 3034 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS which suggests that PhyH-DI has the capacity to increase the catalytic efficiency of PhyH-DII. Further- more, the catalytic efficiency of PhyH was much greater than that of PhyH-DI and PhyH-DII combined (5.73 fold) or twice the amount of PhyH-DII (5.45 fold). This result reveals that the tandemly repeated BPP has higher catalytic efficiency over single-domain phytase, and the incomplete domain might play a key role. Catalytic efficiencies of fusion BPP constructs To verify the above conjecture, two fusion proteins, PhyH-DI-168PhyA and PhyH-DI-PhyP, were con- structed and expressed in E. coli. The catalytic con- stants (k cat ) of PhyH-DI-168PhyA and PhyH-DI-PhyP towards InsP 6 are 12.28 s )1 at 55 °C and 94.4 s )1 at 37 °C, respectively, which are significantly greater than those of the respective wild-type single-domain phyta- ses (Table 1). Discussion In the present study, a BPP gene (phyH) was cloned from Bacillus sp. HJB17, a strain isolated from the alpine tundra soil of China No. 1 Glacier. It has been reported that enzymes produced by psychrophilic organisms are catalytically efficient at low tempera- tures but are less stable at mesophilic temperatures [17,18]. Bacillus sp. HJB17 has optimal growth at 37 °C and is not strictly psychrophilic. The apparent optimal temperature of PhyH is 35 °C, similar to the optimal temperature of strain HJB17, and the optimal pH is 7.0. PhyH has some cold-adaptive properties, retaining 20% of its maximal activity at 0 °C and being thermolabile at 45 °C (Fig. 3). PhyH may be 0 20 40 60 80 100 120 Relative activity (%) 10 m M 5 m M 1 m M 0 m M 0 20 40 60 80 100 120 0 102030405060708090 Relative activity (%) 35 °C20°C 35 °C20°C45°C 45 °C 0 20 40 60 80 100 120 0 102030405060708090 Time at 35 °C (min) Time at 60 °C (min) Time at 60 °C ( min) Time at 45 °C (min) Relative activity (%) 0 20 40 60 80 100 120 0 5 10 15 20 25 30 05 10 15 20 25 30 Relative activity (%) 10 m M 5 m M 1 m M 0 m M AB CD Fig. 3. Thermostability of PhyH determined at 20, 35 and 45 °C after incubation at 35 °C (A) or 45 °C (B), and thermostability of PhyH (C) and PhyH-DII (D) at 60 °C in the presence of 0–10 m M Ca 2+ . –1 –0.5 0 0.5 1 1.5 2 250 260 270 280 290 300 310 320 Wave le ngth (nm) θ (mdeg) Fig. 4. Near-UV CD spectra of PhyH incubated at 35 °C for 0 h (black solid line) and 12 h (black dashed line) or boiled for 5 min (gray solid line) and 2 h after boiling (gray dashed line). Z. Li et al. Catalysis of a dual-domain b-propeller phytase FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3035 used for aquaculture where the temperature is low (28–30 °C) and the pH is neutral [19,20]. Sequence and structural analysis showed that PhyH contains two domains – incomplete PhyH-DI and complete PhyH-DII. Both PhyH and PhyH-DII degrade InsP 6 efficiently, whereas PhyH-DI does not. These observations have also been reported for PhyS and its isolated domains, but the function of its N-ter- minal domain was not characterized [6]. Given that PhyH has a greater catalytic efficiency than does PhyH-DII, we conjecture that PhyH-DI must be involved in InsP 6 hydrolysis. To identify its role in the catalysis process, we tested its substrate specificity and action mode after incubation of InsP 6 with PhyH-DII or other phytases. PhyH-DI can hydrolyze InsP 4 and acts synergistically with other phytases to release 1.2– 2.5-fold phosphate. This is the first time the substrate specificities and functions of the two domains have been characterized in a tandemly repeated BPP. The same phenomenon has been reported for an inositol polyphosphatase PhyAmm, in which the complete D2 domain hydrolyzes the highly phosphorylated IPPs and the incomplete D1 domain targets both IPPs released by the D2 domain and unrelated di-, tri- and tetra-phosphorylated IPPs present in the environment [4,21]. Interestingly, the catalytic activity of PhyH is much greater than the activity sum of PhyH-DI and PhyH-DII and two times greater than that of PhyH- DII. This large variance cannot be ascribed to the function of PhyH-DI alone. The dual-domain phytase was shown to be a dimer according to the native elec- trophoresis. Thus, we presume the intact domain (PhyH-DI) might mediate dimerization and further enhance the catalytic efficiency of the dimer. BPP is widespread in terrestrial and aquatic ecosys- tems. Many putative nucleotide sequences homologous to dual-domain BPP-like phytases are found in the microbial genomes of the NCBI database, e.g. those of Shewanella sp., Pseudomonas sp. and Idiomarina sp. [5], suggesting the prevalence of dual-domain BPPs in c-Proteobacteria. Notably, PhyH is the first Bacillus dual-domain BPP identified, and its dual-domain struc- ture is responsible for its higher catalytic efficiency. This tandemly repeated structure probably evolves from a single domain by gene duplication and persists for better phosphate utilization. Table 1. Kinetic parameters of BPPs and their fused counterparts with PhyH-DI. Enzyme Specific activity (UÆmg )1 ) K m (lM) V max (lmolÆmin )1 Æmg )1 ) k cat (s )1 ) k cat ⁄ K m (lM )1 Æs )1 ) PhyH at 35 °C 4.43 ± 0.55 500 24.82 27.72 0.0540 PhyH at 20 °C 2.81 ± 0.28 1143 15.93 17.76 0.0155 PhyH-DII at 35 °C 1.82 ± 0.23 1432 5.56 4.17 0.0029 PhyH-DI-168PhyA at 55 °C 14.78 ± 0.84 191 10.24 12.28 0.6400 168PhyA at 55 °C 13.02 ± 0.56 240 8.45 5.92 0.2500 PhyH-DI-PhyP at 37 °C 29.82 ± 1.82 1086 78.13 94.40 0.0870 PhyP at 37 °C 21.61 ± 1.36 1280 71.90 44.94 0.0350 Table 2. Phytate hydrolysis by PhyH-DI with other BPPs and HAP. Order of enzyme addition and reaction time Amount of liberated inorganic phosphate (lmol) Folds of increase in activityFirst enzyme Time (min) Second enzyme Time (min) Observed yield Expected yield PhyH-DI 120 – – 0.004 ± 0.003 – – PhyH-DII 120 – – 0.302 ± 0.013 – – PhyH-DII 120 PhyH-DI 120 0.500 ± 0.026 0.313 1.63 168PhyA 5 – – 0.536 ± 0.007 – – 168PhyA 5 PhyH-DI 120 1.345 ± 0.019 0.540 2.49 PhyP 5 – – 0.444 ± 0.008 – – PhyP 5 PhyH-DI 120 0.523 ± 0.014 0.448 1.17 APPA 5 – – 1.978 ± 0.022 – – APPA 5 PhyH-DI 120 2.310 ± 0.004 1.982 1.20 PhyH 120 – – 3.260 ± 0.170 – – PhyH-DI ⁄ DII 120 – – 0.569 ± 0.015 – – 2 · PhyH-DII 120 – – 0.599 ± 0.017 – – Catalysis of a dual-domain b-propeller phytase Z. Li et al. 3036 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS In the present study, we constructed two fusion BPPs that showed greater catalytic constants towards InsP 6 than the original enzymes. It appears that the action of PhyH-DI is general, i.e. not limited to its interaction with PhyH-DII, and can therefore enhance the catalytic efficiencies of single-domain phytases. Thus, PhyH-DI fused to another single-domain phy- tase may improve the catalytic efficiency of the latter. Materials and methods Strains, plasmids and chemicals E. coli Trans1-T1 (TransGen, Beijing, China) and pGEM-T Easy (Promega, Madison, WI, USA) were used for gene cloning and sequencing, respectively. E. coli BL21 (DE3) (TaKaRa, Ostu, Japan) and pET-22b(+) (Novagen, San Diego, CA, USA) were used for heterologous gene expres- sion. T4 DNA ligase and restriction enzymes were supplied by New England Biolabs (Hitchin, Herts, UK). LMW-SDS marker kit and HMW native marker kit were purchased from GE Healthcare (Uppsala, Sweden). Phytate (sodium salt), d-Ins(2)P 1 , d-Ins(1,4)P 2 , d-Ins(1,4,5)P 3 , d-Ins(1,4,5,6)P 4 and Ins(1,3,4,5,6)P 5 were purchased from Sigma (St Louis, MO, USA). Other chemicals were analytical grade and commercially available. Microorganism isolation from glacier soil China No. 1 Glacier (43° 06.1183¢ N, 86° 50.1453¢ E) is located in Xinjiang, China, where the average daily temper- ature is below )20 °C. Samples of alpine tundra soil at an elevation of 3525 m with an eastern exposure were collected in September 2009 and stored at 4 °C. Bacteria were screened for phytase activity at 4, 10, 20, and 37 °C using two types of agar plates: one that contained phytase screen- ing medium [10] and one that contained low phosphate medium [22]. Phytase activity in the supernatants and cell pellets of the retrieved strains was measured using the fer- rous sulfate molybdenum blue method with a small modifi- cation as described below [22,23]. Strain HJB17 with the greatest activity against InsP 6 was identified on the basis of its 16S rDNA gene sequence, and was subjected to further experimentation. Cloning, sequence and structure determination of the phytase gene Strain HJB17 was cultured in Luria–Bertani medium at 37 °C overnight and genomic DNA was extracted using the TIANamp Bacteria DNA kit (Tiangen, Beijing, China). The phytase gene was cloned from the genomic DNA of strain HJB17 with the two-step PCR method according to Huang et al. [10]. Nucleotide sequence assembly was performed using Vec- tor NTI Advance 10.0 software (Invitrogen, Carlsbad, CA, USA), and analyzed using the NCBI ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). DNA and protein sequence alignments used blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/), respectively. The SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) was used for signal peptide analysis. A multiple protein sequence alignment was performed using clustalw (http:// www.ebi.ac.uk/clustalW/) [24]. Protein structure was pre- dicted using swiss-model (htt p://sw issmodel .expasy. org// SWISS-MODEL.html) [25,26] and B. amyloliquefaciens TS- Phy (1POO) [15] as the temp late. Expression and purification of PhyH, PhyH-DI and PhyH-DII in E. coli Three genes phyH, phyH-DI and phyH-DII, the first two lacking contiguous, upstream signal peptide sequences, were each PCR amplified using the expression primers given in Table S1 and were then cloned into the EcoRI–XhoI site of a pET-22b(+) plasmid to construct the recombinant plas- mids (pET-phyH, pET-phyH-DI and pET-phyH-DII; Fig. 5). Each plasmid was transformed into E. coli BL21 (DE3) competent cells. Positive transformants were grown in 25 mL Luria–Bertani medium (pH 7.0), 100 lgÆmL )1 ampicillin at 37 °CtoanD 600 of 0.6. Protein expression was induced by addition of IPTG (1 mm) and Ca 2+ (1 mm) for 20 h at 20 °C. Culture supernatants and cell pellets were assayed for phytase activity and separated by SDS ⁄ PAGE. Purification was performed at 4 °C. The supernatants were concentrated through a hollow fiber cartridge (cutoff 6 kDa; Motianmo, Tianjin, China) and the His-tagged proteins in the supernatants were adsorbed onto a His- Trap HP column (GE Healthcare) following Wang et al. [27]. Purified PhyH was dialyzed against 20 mm Tris ⁄ HCl (pH 7.0), 1 mm Ca 2+ , lyophilized, and dissolved in the same buffer. Native electrophoresis was performed using a non-denaturing 4%–15% (w ⁄ v) polyacrylamide gradient gel at 15 mA at 4 °C. Gels were finally stained with Coo- massie Brilliant Blue R250. Protein concentration was determined using the Bradford assay with BSA as the standard [28]. Phytase activity assay Phytase activity was determined by measuring the amount of phosphate released from InsP 6 using a modified ferrous sulfate molybdenum blue method [23,29]. One unit (U) of phytase activity was defined as the amount of enzyme required to liberate 1 lmol phosphate per minute at the corresponding temperature. All determinations were performed in triplicate. Z. Li et al. Catalysis of a dual-domain b-propeller phytase FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3037 Characterization of purified recombinant PhyH, PhyH-DI and PhyH-DII To determine the effect of Ca 2+ , phytase activity was assayed at 37 °C in solutions of 100 mm Tris ⁄ HCl (pH 7.0) that contained 0–5.0 mm CaCl 2 . The optimal pH for enzyme activity was determined in the presence of the opti- mal Ca 2+ concentration at 37 °C for 30 min with the fol- lowing buffers: 100 mm glycine ⁄ HCl (pH 1.0–3.5), 100 mm sodium acetate ⁄ acetic acid (pH 3.5–6.0), 100 mm Tris ⁄ HCl (pH 6.0–8.5) and 100 mm glycine ⁄ NaOH (pH 8.5–12.0). Phytase activity was measured between 0 and 60 °Cto determine the apparent optimal temperature for activity. The effect of pH on enzyme stability was determined by measuring the residual activity after incubating the enzymes in buffered solutions with pH values of 3.0–10.0 at 37 °C for 1 h. The thermostability of PhyH at 35 and 45 °C was deter- mined by measuring the residual activity at 20, 35 or 45 ° C after incubation for various periods of time in 100 mm Tris ⁄ HCl (pH 7.0). Additionally, the thermostabilities of PhyH and PhyH-DII were also investigated at 60 °C in the presence of 0–10 mm Ca 2+ . An enzyme solution without treatment served as the control and was considered to have 100% activity. Kinetic parameters for InsP 6 activity were determined in 100 mm Tris ⁄ HCl (pH 7.0) containing 1 mm Ca 2+ and 0.0125–2 mm InsP 6 . Reactions were run for 5 min at 35 °C and 20 °C, respectively. The K m and V max values were determined using Lineweaver–Burk plots [30] and the non- linear regression computer program grafit. Three indepen- dent experiments were averaged, and each experiment included three replicates. Circular dichroism spectroscopy To verify the structural stability of PhyH at 35 °C or after boiling, near-UV CD signals between 250 and 320 nm were measured with a MOS-450 CD spectrometer (Bio-Logic, Claix, France) equipped with a TCU-250 Peltier-type tem- perature control system. PhyH at a concentration above 1mgÆmL )1 in a 10-mm cell was subjected to signal mea- surement within 8 min. PhyH-DI substrate specificity The substrate specificity of PhyH-DI was determined by measuring its activity after incubation in 100 mm Tris ⁄ HCl (pH 7.0) with 1 mmd-Ins(2)P 1 , d-Ins(1,4)P 2 , d-Ins(1,4,5)P 3 , d-Ins(1,4,5,6)P 4 , Ins(1,3,4,5,6)P 5 or InsP 6 at 37 °C for 30 min. Each assay was replicated three times. The function of PhyH-DI in InsP 6 hydrolysis degradation To determine the role of PhyH-DI in InsP 6 hydrolysis, its effect on the enzymatic activities of PhyH-DII, 168PhyA [9], PhyP [10] and E. coli AppA was characterized in a two- step process. Each sample containing 1.5 mm sodium phy- tate and 25 nmol of one of the phytases listed above, in 100 mm Tris ⁄ HCl (pH 6.0 or 7.0) for BPPs or in 100 mm sodium acetate (pH 5.0) for AppA, was incubated at 37 °C for 5 or 120 min and boiled for 5 min to inactivate the enzyme present. Then the samples were adjusted to pH 7.0, 25 nmol PhyH-DI was or was not added, and the samples were incubated at 37 °C for 120 min. The reactions were terminated by addition of 1.5 mL trichloroacetic acid PhyP-PhyDI-R PhyDI-PhyP-F PhyH-F Phy168-R PhyH-F PhyH-R PhyP-R PhyDI-Phy168-F phyH-DII PhyH-F PhyDI-R PhyDII-F PhyH-R PhyH-F Phy168-PhyDI-R phyH-DI 168phyA phyH-DII phyH-DI phyH-DI phyP phyH-DI pET-phyH pET-phyH-DI pET-phyH-DI-phyP p ET-phyH-DI-168phyA pET-phyH-DI Fig. 5. Expression plasmid construction. The arrows indicate the locations and directions of the PCR primers. Catalysis of a dual-domain b-propeller phytase Z. Li et al. 3038 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS (10%, w ⁄ v) and subjected to the ferrous sulfate molybde- num blue assay. Samples that contained only PhyH-DI or one of the other phytases served as controls. To understand how the two PhyH domains interact dur- ing catalysis, samples that contained 25 nmol PhyH, a com- bination of 25 nmol PhyH-DI and 25 nmol PhyH-DII, or 50 nmol PhyH-DII and 1.5 mm sodium phytate in 100 mm Tris ⁄ HCl (pH 7.0) were incubated at 37 °C for 120 min, terminated by the addition of 1.5 mL trichloroacetic acid, and subjected to the ferrous sulfate molybdenum blue assay. Construction, expression and characterization of fused BPPs The fusion genes phyH-DI-phyP and phyH-DI-168phyA, with phyH fused upstream, were constructed by overlapping PCR [31] with the primers given in Table S1. EcoRI and XhoI cleavage sites were introduced into the 5¢ end of phyH-DI and the 3¢ end of phyP or 168phyA, respectively. Each gene was restriction digested and inserted into pET- 22b(+). Expression, purification and characterization of the two fusion proteins were conducted as described above. Acknowledgements This work was supported by the National Natural Sci- ence Foundation of China (31001025), the Key Pro- gram of Transgenic Plant Breeding (2008ZX08011-005) and the earmarked fund for China Modern Agriculture Research System (CARS-42). The authors declare there is no conflict of interest in this paper. References 1 Reddy NR, Sathe SK & Salunkhe DK (1982) Phytates in legumes and cereals. Adv Food Res 28, 1–92. 2 Mullen MD (2005) Phosphorus in soils: biological inter- actions. In Encyclopedia of Soils in the Environment (Hillel D ed), Vol. 3, pp. 210–215. Elsevier, Oxford, UK. 3 Rao D, Rao KV, Reddy TP & Reddy VD (2009) Molecular characterization, physicochemical properties, known and potential applications of phytases: an over- view. Crit Rev Biotechnol 29, 182–198. 4 Huang H, Shi P, Wang Y, Luo H, Shao N, Wang G, Yang P & Yao B (2009) Diversity of b-propeller phytase genes in the intestinal contents of grass carp provides insight into the release of major phosphorus from phytate in nature. Appl Environ Microbiol 75, 1508–1516. 5 Lim BL, Yeung P, Cheng C & Hill JE (2007) Distribu- tion and diversity of phytate-mineralizing bacteria. ISME J 1, 321–330. 6 Cheng C & Lim BL (2006) b-Propeller phytases in the aquatic environment. Arch Microbiol 185, 1–13. 7 Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N & Apajalahti J (1998) Isolation, characterization, molecu- lar gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 64, 2079– 2085. 8 Kim Y, Lee J, Kim H, Yu J & Oh T (1998) Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiol Lett 162, 185–191. 9 Tye AJ, Siu FK, Leung TYC & Lim BL (2002) Molecu- lar cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. Appl Microbiol Biotechnol 59 , 190–197. 10 Huang H, Shao N, Wang Y, Luo H, Yang P, Zhou Z, Zhan Z & Yao B (2009) A novel b-propeller phytase from Pedobacter nyackensis MJ11 CGMCC 2503 with potential as an aquatic feed additive. Appl Microbiol Biotechnol 83, 249–259. 11 Zhang R, Yang P, Huang H, Yuan T, Shi P, Meng K & Yao B (2011) Molecular and biochemical character- ization of a new alkaline b -propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115. Appl Microbiol Biotechnol doi: 10.1007/s00253- 011-3309-0. 12 Fu S, Sun J & Qian L (2008) Effect of Ca 2+ on b-pro- peller phytases. Protein Pept Lett 15, 39–42. 13 Kim OH, Kim YO, Shim JH, Jung YS, Jung WJ, Choi WC, Lee H, Lee SJ, Auh JH & Kim KK (2010) b-Pro- peller phytase hydrolyzes insoluble Ca 2+ -phytate salts and completely abrogates the ability of phytate to chelate metal ions. Biochemistry 49, 10216–10217. 14 Kerovuo J, Rouvinen J & Hatzack F (2000) Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism. Biochem J 352, 623–628. 15 Ha NC, Oh BC, Shin S, Kim HJ, Oh TK, Kim YO, Choi KY & Oh BH (2000) Crystal structures of a novel, thermostable phytase in partially and fully calcium- loaded states. Nat Struct Mol Biol 7, 147–153. 16 Cheng C, Wong KB & Lim BL (2007) The effect of disulfide bond on the conformational stability and catalytic activity of b-propeller phytase. Protein Pept Lett 14, 175–183. 17 Collins T, Meuwis MA, Gerday C & Feller G (2003) Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. J Mol Biol 328, 419–428. 18 D’Amico S, Collins T, Marx JC, Feller G & Gerday C (2006) Psychrophilic microorganisms: challenges for life. EMBO Rep 7, 385–389. 19 Cao L, Wang W, Yang C, Yang Y, Diana J, Yakupitiy- age A, Luo Z & Li D (2007) Application of microbial phytase in fish feed. Enzyme Microb Tech 40, 497–507. Z. Li et al. Catalysis of a dual-domain b-propeller phytase FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3039 20 Mullaney EJ, Daly CB & Ullah AHJ (2000) Advances in phytase research. Adv Appl Microbiol 47, 157–199. 21 Gruninger RJ, Selinger LB & Mosimann SC (2009) Struc- tural analysis of a multifunctional, tandemly repeated inositol polyphosphatase. J Mol Biol 392, 75–86. 22 Lei XG, Porres JM, Mullaney EJ & Brinch-Pedersen H (2007) Phytase: source, structure and application. In Industrial Enzymes: Structure, Function and Applications (Polaina J & MacCabe AP eds), pp 505–529. Springer, Dordrecht. 23 Holman WI (1943) A new technique for the determina- tion of phosphorus by the molybdenum blue method. Biochem J 37, 256–259. 24 Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weight- ing, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680. 25 Arnold K, Bordoli L, Kopp J & Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformat- ics 22, 195–201. 26 Guex N & Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18 , 2714–2723. 27 Wang G, Luo H, Wang Y, Huang H, Shi P, Yang P, Meng K, Bai Y & Yao B (2011) A novel cold-active xylanase gene from the environmental DNA of goat rumen contents: direct cloning, expression and enzyme characterization. Bioresour Technol 102, 3330–3336. 28 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254. 29 Makarewicz O, Dubrac S, Msadek T & Borriss R (2006) Dual role of the PhoP approximately P response regulator: Bacillus amyloliquefaciens FZB45 phytase gene transcription is directed by positive and negative interactions with the phyC promoter. J Bacteriol 188, 6953–6965. 30 Lineweaver H & Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56, 658–666. 31 Horton R, Hunt H, Ho S, Pullen J & Pease L (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68. Supporting information The following supplementary material is available: Fig. S1. clustalw alignment of the PhyH-DI and PhyH-DII amino acid sequences with those of the clo- sely related BPPs, including Shewanella oneidensis MR- 1 PhyS-DI and PhyS-DII [6], Bacillus amyloliquefaciens TS-Phy [15], Bacillus subtilis 168 168PhyA [9] and Pe- dobacter nyackensis MJ11 PhyP [10]. Table S1. Primers used in this study. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Catalysis of a dual-domain b-propeller phytase Z. Li et al. 3040 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS . The tandemly repeated domains of a b-propeller phytase act synergistically to increase catalytic efficiency Zhongyuan Li*, Huoqing Huang*, Peilong Yang,. evolutionarily because they can increase the amount of available phosphate by interacting together. Additionally, fusing PhyH-DI to a single-domain phytase appears