The pathway of dephosphorylation of myo-inositol hexakisphosphate ...

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The pathway of dephosphorylation of myo-inositol hexakisphosphate by phytate-degrading enzymes of different Bacillus spp. Ralf Greiner, Adelazim Farouk, Marie Larsson Alminger, and Nils-Gunnar Carlsson

Abstract: The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies. The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3. The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration. Key words: Bacillus spp., myo-inositol phosphate isomers, phytase, phytate degradation. Résumé : Nous avons reconstitué la voie de déphosphorylation du myo-inositol hexakisphosphate Greiner par et al. les enzymes dégradant le phytate qu’on retrouve chez Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, et Bacillus amyloliquefaciens 45 à l’aide d’analyses de chromatographie ionique à haute performance et d’études de cinétique. Les données démontrent que toutes les enzymes de Bacillus étudiées dégradant le phytate pouvaient déphosphoryler le myoinositol hexakisphosphate par l’enlèvement séquentiel de groupes phosphate et ce, par le biais de deux voies indépendantes: les voie passent par le D-Ins(1,2,4,5,6)P5, le Ins(2,4,5,6)P4 pour aboutir au Ins(2,4,6)P3 ou au D-Ins(2,5,6)P3, et par le D-Ins(1,2,4,5,6)P5, le D-Ins(1,2,5,6)P4 pour aboutir au D-Ins(1,2,6)P3. Le myo-inositol trisphosphate D-Ins(1,2,6)P3 résultant a été dégradé en du Ins(2)P en passant par le D-Ins(2,6)P2, à la suite de temps d’incubation prolongés combinés à une concentration plus élevée d’enzyme. Mots clés : Bacillus spp., isomères de myo-inositol phosphate, phytase, dégradation du phytate. [Traduit par la Rédaction]

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Introduction Phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. Phytate-degrading enzymes were originally proposed as an animal feed additive to enhance the value of plant material in animal feed by liberating orthophosphate (Ravindran et al. 1995). More recently, addition of phytate-degrading enzymes has also been seen as a way Received 5 April 2002. Revision received 18 October 2002. Accepted 22 October 2002. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 5 December 2002. R. Greiner.1 Centre for Molecular Biology, Federal Research Centre for Nutrition, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany. A. Farouk. Department for Biology, Humboldt University, Chausseestrasse 117, 10115 Berlin, Germany. M.L. Alminger and N.-G. Carlsson. Department of Food Science, Chalmers University of Technology, c/o SIK, Box 5401, SE-402 29 Göteborg, Sweden. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 48: 986–994 (2002)

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to reduce the level of phosphate pollution in areas of intensive lifestock management (Cromwell et al. 1995; Simons et al. 1990). As phytate can act as an antinutrient by binding to proteins and by chelating minerals, such as zinc, iron, calcium, and magnesium (Cheryan 1980), addition of phytatedegrading enzymes can improve the nutritional value of plant-based foods by enhancing protein digestibility and mineral availability through phytate hydrolysis during digestion in the stomach (Sandberg et al. 1996; Yi and Kornegay 1996) or during food and feed processing (Reddy et al. 1989). In recent years, phytate-degrading enzymes were also of interest for producing defined breakdown products of phytate for kinetic and physiological studies. The physiological role of different myo-inositol phosphates is presently undergoing extensive research. Certain myo-inositol phosphates have been proposed to have novel metabolic effects, such as amelioration of heart disease (Jariwalla et al. 1990; Potter 1995), prevention of renal stone formation (Modlin 1980; Ohkawa et al. 1984), and reduction in the risk of colon cancer (Baten et al. 1989; Graf and Eaton 1993; Shamsuddin et al. 1997; Ullah and Shamsuddin 1990; Vucenik et al. 1993; Yang and Shamsuddin 1995). Furthermore, D-myo-inositol(1,2,6)trisphosphate has been studied with respect to the prevention of dia-

DOI: 10.1139/W02-097

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betes complications and treatment of chronic inflammations, as well as cardiovascular diseases (Carrington et al. 1993; Claxon et al. 1990; Ruf et al. 1991; Siren et al. 1991). In addition, much attention has been focused on lower myoinositol phosphates, in particular the intracellular second messengers D-myo-inositol(1,4,5)trisphosphate and D-myoinositol(1,3,4,5)tetrakisphosphate, which affect cellular metabolism and secretion by stimulating intracellular release of calcium (Potter 1990). Different phytate-degrading enzymes may result in different positional isomers of the lower myoinositol phosphates and, therefore, in different physiological effects. However, the complete hydrolysis pathway of phytate by phytate-degrading enzymes has been elucidated in rare cases only, such as for the phytate-degrading enzymes from spelt, rye, barley, oat (Greiner and Larsson Alminger 2001), wheat (Lim and Tate 1973; Nakano et al. 2000), rice (Hayakawa et al. 1990), lupine (LP11, LP12, LP2) (Greiner 2001), mung bean (Maiti et al. 1974), lily pollen (Barrientos et al. 1994), Saccharomyces cerevisiae (Greiner et al. 2001), Pseudomonas (Cosgrove 1970), Paramecium (van der Kaay and van Haastert 1995), Escherichia coli (Greiner et al. 2000), and Bacillus subtilis (Kerovuo et al. 2000). Difficulties in separating isomers of myo-inositol phosphates have been reported in connection with several analytical approaches. During the last few years, a number of isomer-specific, ion-exchange chromatography methods have been developed (Mayr 1988; Phillippy and Bland 1988; Skoglund et al. 1997, 1998). Remaining problems, however, are the separation of isomers from the whole spectrum of myo-inositol phosphates in the same run and the missing availability of several myo-inositol phosphates as reference compounds. In addition, the 24 enantiomeric pairs among the myo-inositol phosphate isomers are not separated on the achiral columns in use. Therefore, the absolute configurations of such enantiomers have to be determined using high-affinity binding proteins or enzymatic assays (Greiner and Larsson Alminger 2001; Greiner et al. 2000, 2001). In this paper, we report the dephosphorylation of phytate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45.

Materials and methods Chemicals Aspergillus niger phytase was obtained from Novo Nordisk (Copenhagen, Denmark). Phytic acid dodecasodium salt was from Aldrich (Steinheim, Germany). Ultrasep ES 100 RP18 was purchased from Bischoff (Leonberg, Germany) and highperformance ion chromatography (HPIC) Carbo-Pac PA-100 column from Dionex (Sunnyvale, Calif., U.S.A.). AG1 X-4, 100–200 mesh resin was obtained from Bio-Rad (München, Germany). Purification of the phytate-degrading enzymes Purification of the phytate-degrading enzymes of A. niger (Greiner et al. 2001), E. coli (Greiner et al. 1993), and rye (Secale cereale) (Greiner et al. 1998) was performed as described previously. The genes encoding phytate-degrading enzymes from the different Bacillus strains (B. subtilis 168: accession Number

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P42094, Swiss Prot Database; B. amyloliquefaciens ATCC 15841: accession Number AF453255, National Center for Biotechnology Information (NCBI) Database; B. amyloliquefaciens 45: accession Number AY055220, NCBI Database) were overexpressed in B. subtilis MU331 using the vector pSG1112 (Thornewell et al. 1993). Purification of the phytate-degrading enzymes secreted into the medium was achieved by dialysing the culture supernatant against 20 mM sodium acetate buffer, pH 5.0. The dialysates were applied onto a Mono S HR 5/5 column equilibrated with 20 mM sodium acetate buffer, pH 5.0. The column was first washed with the same buffer for 30 min, and then a linear gradient consisting of 0–0.5 M NaCl in 20 mM sodium acetate buffer, pH 5.0, for 60 min was applied. Two-millilitre fractions were collected. The fractions containing phytatedegrading activity were pooled, dialysed against 20 mM Tris–HCl buffer, pH 7.5, and applied onto a Mono Q HR 5/5 column equilibrated with 20 mM Tris–HCl buffer, pH 7.5. The column was first washed with the same buffer for 30 min, and then a linear gradient consisting of 0–0.5 M NaCl in 20 mM Tris–HCl buffer, pH 7.5, for 30 min was applied. Two-millilitre fractions were collected. The fractions containing phytate-degrading activity were pooled. Both columns were run at 25°C and a flow rate of 1 mL min–1. All phytate-degrading enzymes were purified to apparent homogeneity according to denaturing and nondenaturing polyacrylamide gel electrophoresis. Native gel electrophoresis was carried out with 5% gels at pH 4.8 (Ornstein 1964). Enzymatic staining of the protein was performed with 1-naphthyl phosphate coupled with fast blue B in 0.1 M Tris–HCl, 2 mM CaCl2, pH 7.5, in the dark (Dorn 1965). SDS-electrophoresis using 10% gels was performed according to Laemmli (1970). Gels were stained by Coomassie brilliant blue R-250. Assay of phytate-degrading activity Phytate-degrading activity measurements were carried out at 37°C. The enzymatic reactions were started by the addition of 10 µL enzyme to the assay mixtures. The incubation mixture for phytate-degrading activity determination consisted of 350 µL 0.1 M sodium acetate, pH 4.5 (E. coli, A. niger), 0.1 M sodium acetate, pH 6.0 (rye), and 0.1 M Tris– HCl, 2 mM CaCl2, pH 7.5 (Bacillus spp.), respectively, containing 500 nmol sodium phytate. To determine the kinetic parameters for enzymatic dephosphorylation of individual myo-inositol phosphates by the phytate-degrading enzymes under investigation, 10 mU of the enzymes were added to sequentially diluted solutions (2.0, 1.0, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015 mM) of the purified myo-inositol phosphate isomers (D-Ins(1,2,4,5,6)P5, DIns(1,2,3,4,5)P5, Ins(2)P, D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, and the myo-inositol pentakisphosphate produced by the phytate-degrading enzyme under investigation) in 400 µL 0.1 M Tris–HCl, 2mM CaCl2, pH 7.0, at 37°C. After an incubation period of 30 min, the liberated phosphate was quantified by a modification of the ammonium molybdate method (Heinonen and Lahti 1981). The rate of reaction was linear for the 30-min incubation time (data not shown). Activity (U) was expressed as micromoles of phosphate liberated per minute. Blanks were run by the addition of the ammonium molybdate solution prior to addition of the © 2002 NRC Canada



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enzyme solution to the assay mixture. The kinetic constants (Km, Vmax) were calculated from Lineweaver–Burk plots of the data. A molecular mass of 40 kDa was used for the calculation of kcat. Quantification of the liberated phosphate A freshly prepared solution (1.5 mL) of acetone: 2.5 M sulfuric acid : 10 mM ammonium molybdate (2:1:1, v/v), and thereafter, 100 µL 1.0 M citric acid were added to 400 µL of the suitably diluted hydrolysis mixtures or the mixtures of the phytase assay. Any cloudiness was removed by centrifugation (10 000 × g at 20°C for 5 min) prior to the measurement of absorbance at 355 nm. To quantify the released phosphate, a calibration curve was produced over the range of 5–600 nmol phosphate. Preparation of lower myo-inositol phosphates The phytate-degrading enzymes from A. niger, E. coli, and rye were used to generate D-Ins(1,2,4,5,6)P5, D-Ins(1,2,3,4,5,)P5, D-Ins(1,2,3,5,6)P5, D-Ins(1,2,5,6)P4, and D-Ins(1,2,6)P3. Myoinositol hexakisphosphate (500 µmol) was incubated at 37°C in a mixture containing 50 mM NH4-acetate, pH 4.5 (E. coli, A. niger), 50 mM NH4-acetate, pH 6.0 (rye), and 50 mM Tris–HCl, 2 mM CaCl2, pH 7.5 (Bacillus spp.), and 10 U of the phytate-degrading enzymes in a final volume of 200 mL. After an incubation period of 30 min (InsP5-production) and 90 min (InsP4- and InsP3-production), respectively, the reactions were stopped by heat treatment (95°C, 10 min). The incubation mixtures were lyophilised, and the dry residues were dissolved in 10 mL 0.2 M NH4-formate, pH 2.5. The solutions were loaded onto a Q-Sepharose column (2.6 × 110 cm) (Pharmacia, Freiburg, Germany) equilibrated with 0.2 M NH4-formate, pH 2.5, at a flow rate of 2.5 mL min–1. The column was washed with 600 mL of 0.2 M NH4-formate, pH 2.5. The bound myo-inositol phosphates were eluted stepwise using 600 mL 0.6 M NH4-formate, pH 2.5; 1200 mL 0.8 M NH4-formate, pH 2.5; and 600 mL 1.0 M NH4-formate, pH 2.5, at a flow rate of 2.5 mL min–1. Fractions of 10 mL were collected. From even-numbered tubes, 100-µL aliquots were lyophilised. The residues were dissolved in 1.5 M sulfuric acid and incubated for 90 min at 165°C to hydrolyse the eluted myo-inositol phosphates completely. The liberated phosphate was quantified by a modification of the ammonium molybdate method (Heinonen and Lahti 1981). The content of the fraction tubes corresponding to the individual myo-inositol phosphates were pooled and lyophilised until only a dry residue remained. Ten millilitres of water were used to redissolve the residues. Lyophilisation and redissolving were repeated twice. Myo-inositol phosphate concentrations were determined by HPLC ion-pair chromatography on Ultrasep ES 100 RP18 (2 × 250 mm). The column was run at 45°C and 0.2 mL min–1 with an eluant consisting of formic acid:methanol: water:tetrabutyl-ammonium hydroxide (44:56:5:1.5, v/v), pH 4.25, as described by Sandberg and Ahderinne (1986). A mixture of the individual myo-inositol phosphate esters (InsP3–InsP6) was used as a standard. The purity of the myo-inositol phosphate preparations was determined on a HPIC system as described by Skoglund et al. (1998). Enzymatic phytate degradation The enzymatic reaction was started at 37°C by the addi-

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tion of 50 µL of the suitable diluted solution of the phytatedegrading enzyme under investigation to the incubation mixtures (100 mU mL–1). The incubation mixture consisted of 1250 µL 0.1 M Tris–HCl, pH 7.5, containing 2.5 µmol CaCl2 and 2.5 µmol sodium phytate. From the incubation mixture, 100-µL samples were removed periodically, and the reaction was stopped by heat treatment (90°C, 5 min). Identification of enzymatically formed hydrolysis products (InsP6–InsP2) The heat-treated samples (50 µL) were chromatographed on a HPIC system using a Carbo-Pac PA-100 (4 × 250 mm) analytical column and a gradient of 5–98% HCl (0.5 M, 0.8 mL min–1) as described by Skoglund et al. (1998). The eluants were mixed in a post-column reactor with 0.1% Fe(NO3)3·9H2O in a 2% HClO4 solution (0.4 mL min–1), according to Phillippy and Bland (1988). The combined flow rate was 1.2 mL min–1. Identification of the myo-inositol monophosphate isomer Myo-inositol monophosphates were produced by incubation of 1.0 U of the phytate-degrading enzyme under investigation with a limiting amount of myo-inositol hexakisphosphate (0.1 µmol) in a final volume of 500 µL of 50 mM NH4-formate. After lyophilisation, the residues were dissolved in 500 µL of a solution of pyridine:bis(trimethylsilyl)trifluoroacetamide (1:1, v/v) and incubated at room temperature for 24 h. The silylated products were injected into a gas chromatograph coupled with a mass spectrometer at 270°C. The stationary phase was methylsilicon in a fused silica column (0.25 mm × 15 m). Helium was used as the carrier gas at a flow rate of 0.5 m s–1. The following heating program was used for the column: 100–340°C, rate increase; 4°C min–1. Ionization was performed by electron impact at 70 eV and 250°C. Statistical methods The Student’s t test was used for statistical comparison.

Results Intermediates of enzymatic myo-inositol hexakisphosphate dephosphorylation The identification of the hydrolysis products of myoinositol hexakisphosphate generated by the phytate-degrading enzymes from B. subtilis 168, B. amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 was performed by isomer-specific HPIC analysis. All three enzymes behave very similar in phytate degradation. Therefore, only the result of B. amyloliquefaciens ATCC 15841 is shown in Fig. 1. The chromatographic profile of the zero-time control indicated only the Ins(1,2,3,4,5,6)P 6 peak. After 10 min incubation, the quantity of Ins(1,2,3,4,5,6)P6 had decreased, and D/LIns(1,2,4,5,6)P5 appeared as the major degradation product, accompanied by small amounts of D/L-Ins(1,2,3,4,5)P5, Ins(2,4,5,6)P4, and D/L-Ins(1,2,5,6)P4. Furthermore, traces of D/L-Ins(1,2,4,6)P4 were found. After 30 min, a further decrease of Ins(1,2,3,4,5,6)P6 was observed, and Ins(2,4,5,6)P4 as well as D/L-Ins(1,2,5,6)P4 had increased. No significant change was found in the quantity of D/L-Ins(1,2,4,5,6)P5 and D/L-Ins(1,2,4,6)P4, while D/L-Ins(1,2,3,4,5)P5 had decreased. After 60 min, Ins(1,2,3,4,5,6)P6, D/L-Ins(1,2,4,5,6)P5, and D/L© 2002 NRC Canada



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Fig. 1. HPIC analysis of hydrolysis products of myo-inositol hexakisphosphate by a purified phytate-degrading enzyme from Bacillus amyloliquefaciens ATCC 15841.(A) Reference sample. The source of the reference myo-inositol phosphates is as indicated in Skoglund et al. (1997, 1998). The phytate-degrading enzyme from B. amyloliquefaciens ATCC 15841 for the following incubation times are shown: (B) 10 min; (C) 30 min; (D) 60 min; (E) 120 min, threefold enzyme concentration; (F) 300 min, tenfold enzyme concentration. Peaks: (A) Ins(1,3,4,5,6)P5; (B) Ins(1,2,3,4,6)P5; (C) D/L-Ins(1,4,5,6)P4; (D) D/L-Ins(1,3,4,5)P4; (E) D/L-Ins(1,2,4,5)P4; (F) Ins(1,3,4,6)P4; (G) D/L-Ins(1,2,3,4)P4; (H) Ins(1,2,3,5)P4; (K) Ins(4,5,6)P3; (L) D/L-Ins(1,5,6)P3; (M) D/L-Ins(1,4,5)P3; (N) D/L-Ins(1,3,4)P3; (O) D/LIns(1,2)P2, Ins(2,5)P2, D/L-Ins(4,5)P2; (P) D/L-Ins(1,4)P2, D/L-Ins(1,6)P2.

Ins(1,2,3,4,5)P5 were completely degraded to Ins(2,4,5,6)P4, D/L-Ins(1,2,5,6)P4, D/L-Ins(1,2,6)P3, and D/L-Ins(2,4,5)P3. After 90 min, only myo-inositol trisphosphate peaks (D/L-Ins(1,2,6)P3 and D/L-Ins(2,4,5)P3) remained (data not shown). Increasing the enzyme concentration threefold and the incubation time to 120 min produced a small quantity of D/LIns(2,4)P2. A further increase in enzyme concentration to tenfold of the original value and a prolonged incubation time of 300 min resulted in a nearly complete degradation of D/LIns(1,2,6)P3 to D/L-Ins(2,4)P2, whereas no significant change in D/L-Ins(2,4,5)P3 was observed. Incubation of the Bacillus phytate-degrading enzymes under investigation with a limiting amount of phytate resulted in the generation of a myoinositol monophosphate, which was identified as Ins(2)P by gas chromatography–mass spectrometry (data not shown).

Kinetic studies The kinetic parameters for the enzymatic degradation of the myo-inositol phosphates studied (Ins(1,2,3,4,5,6)P6, DIns(1,2,4,5,6)P5, D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,4,5)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, Ins(2)P, and the major myoinositol pentakisphosphate generated by the enzyme under investigation) were not statistically different among the phytatedegrading enzymes under investigation (Table 1). Km and kcat for the enzymatic hydrolysis of Ins(1,2,3,4,5,6)P6 were determined to be ~435 µmol L–1 and ~18.6 s–1, respectively. In comparison with myo-inositol hexakisphosphate, the affinity of the four myo-inositol pentakisphosphates for the phytatedegrading enzymes and their maximal rates of hydrolysis were lower. Km and kcat for the enzymatic hydrolysis of DIns(1,2,4,5,6)P5 and the myo-inositol pentakisphosphate inter© 2002 NRC Canada



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Can. J. Microbiol. Vol. 48, 2002 Table 1. Kinetic constants for enzymatic myo-inositol phosphate dephosphorylation. Km (µmol L–1) / kcat (s–1) Substrate

B. amyloliquefaciens ATCC 15841

B. amyloliquefaciens 45

B. subtilis 168

InsP6 InsP5* D-Ins(1,2,4,5,6)P5 D-Ins(1,2,3,5,6)P5 D-Ins(1,2,3,4,5)P5 D-Ins(1,2,5,6)P4 D-Ins(1,2,6)P3 InsP3* Ins(2)P

442±26 / 18.5±0.5c 502±30 / 16.2±0.3a 491±25 / 16.0±0.4a 757±16 / 7.3±0.1b 773±21 / 7.5±0.2b 542±13 / 10.1±0.3d 997±27 / 1.9±0.2e 2461±56 / 0.18±0.02f 4115±87 / 0.12±0.01g

429±28 / 18.7±0.7c 512±20 / 16.5±0.2a 511±29 / 16.2±0.4a 739±19 / 7.6±0.2b 751±26 / 7.4±0.4b 556±17 / 10.4±0.5d 1012±29 / 1.7±0.1e 2517±81 / 0.21±0.04f 4196±79 / 0.14±0.02g

433±21 / 18.8±0.4c 496±27 / 15.9±0.3a 504±19 / 16.1±0.2a 761±21 / 7.2±0.3b 769±17 / 7.4±0.5b 539±19 / 10.8±0.4b 1006±31 / 1.8±0.2e 2493±71 / 0.17±0.03f 4161±94 / 0.11±0.01g

Note: B. amyloliquefaciens, Bacillus amyloliquefaciens; B. subtilis, Bacillus subtilis. The data are mean values ± standard deviation of five independent experiments. Means accompanied by a different letter are significantly different (P < 0.05). *Generated by the phytate-degrading enzyme under investigation.

mediates generated by the phytate-degrading enzymes under investigation were found to be ~500 µmol L–1 and ~16 s–1, respectively, whereas Km and kcat for the enzymatic degradation of D-Ins(1,2,3,5,6)P5 and D-Ins(1,2,3,4,5)P5 were determined to be ~760 µmol L–1 and 7.4 s–1, respectively. Ins(2)P and the mixture of myo-inositol trisphosphate intermediates generated by the phytate-degrading enzymes under investigation were hydrolysed very poorly as reflected by the extremely low kcat of ~0.12 s–1 and ~0.18 s–1, respectively. myo-inositol tetrakisphosphates generated by myoinositol pentakisphosphate dephosphorylation The identification of the myo-inositol tetrakisphosphates was performed by isomer-specific HPIC analysis (data not shown). We analysed the myo-inositol tetrakisphosphates that were generated by the action of the phytate-degrading enzymes on the available myo-inositol pentakisphosphates that were identified as possible intermediates of phytate dephosphorylation (D-Ins(1,2,4,5,6)P5, D-Ins(1,2,3,4,5)P5, and L-Ins(1,2,3,4,5)P5 (identical with D-Ins(1,2,3,5,6)P5)). Degradation of D-Ins(1,2,4,5,6)P5 resulted in the formation of Ins(2,4,5,6)P4 and D-Ins(1,2,5,6)P4 with all three Bacillus enzymes, whereas D-Ins(1,2,3,4,5)P5 and D-Ins(1,2,3,5,6)P5 were degraded to Ins(1,2,3,5)P4.

Discussion Highly thermostable phytate-degrading enzymes have been isolated and cloned from Bacillus species (Kerovuo et al. 1998; Kim et al. 1998). These enzymes are virtually identical. They contain 383 amino acid residues with over 90– 98% sequence identity (Borriss and Farouk 2001; Farouk and Borriss 2000; Farouk et al. 2001; Kerovuo 1997; Kim et al. 1997; Park 2000; Yao 2000). The amino acid sequence of these phytate-degrading enzymes do not have homology to the sequences of any other phosphatase listed in the databases, nor do they contain the conserved active-site motifs RHGxRxP and HD found in histidine acid phytate-degrading enzymes. The phytate-degrading enzyme from B. amyloliquefaciens was shown to represent the first example of a six-bladed propeller exhibiting phosphatase activity (Shin et al. 2001). Thus, the Bacillus enzymes represent a novel class

of phosphatases, and in addition, a novel mode of phytate degradation was proposed for the phytate-degrading enzyme from Bacillus sp. (Kerovuo et al. 2000; Shin et al. 2001). Two classes of phytate-degrading enzymes are recognised by the International Union of Pure and Applied Chemistry and the International Union of Biochemistry (IUPAC–IUB); 3-Phytase (EC 3.1.3.8) initially removes orthophosphate from the D-3 (L-1) position of phytate, whereas 6-phytase (EC 3.1.3.26) preferentially initiates the phytate dephosphorylation at the L-6 (D-4) position of the myo-inositol ring. It was concluded that the myo-inositol pentakisphosphate intermediate generated by the phytate-degrading enzymes under investigation is D-Ins(1,2,4,5,6)P5, since the kinetic constants for the degradation of the myo-inositol pentakisphosphate intermediate generated by these enzymes and D-Ins(1,2,4,5,6)P5 are almost identical (Table 1). This conclusion was further confirmed by the observation that the phytate-degrading enzyme from A. niger degrades D-Ins(1,2,4,5,6)P5, the only myoinositol pentakisphosphate intermediate generated by this enzyme (Irving and Cosgrove 1972), with almost identical kinetic constants as the myo-inositol pentakisphosphate intermediates generated by the enzymes under investigation (Table 2). It is very unlikely that L-Ins(1,2,4,5,6)P5, the other possible intermediate according to HPIC, is hydrolysed with the same kinetic constants by the A. niger enzyme. Since the phytate-degrading enzymes under investigation generate DIns(1,2,4,5,6)P5 as the major myo-inositol pentakisphosphate intermediate, they are 3-phytases. The myo-inositol phosphate intermediates generated by the phytate-degrading enzymes under investigation are consistent with the degradation pathway shown in Fig. 2. The enzymes remove phosphate stepwise from the phytate molecule, whereby each myo-inositol intermediate is released from the enzyme and may become a substrate for further hydrolysis. D-Ins(1,2,4,5,6)P5 serves as the starting point for a dual degradation pathway. The final product of pathway 1 is Ins(2)P, which is generated from D-Ins(1,2,4,5,6)P5 via DIns(1,2,5,6)P4, D-Ins(1,2,6)P3, and D-Ins(2,6)P2. Pathway 2 results in the formation of D/L-Ins(2,4,5)P3 via Ins(2,4,5,6)P4. Since all theoretically existing myo-inositol pentakis- and tetrakisphosphate isomers are well resolved on the HPIC system (Fig. 1), the identity of the myo-inositol pentakis© 2002 NRC Canada



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Table 2. Kinetic constants for myo-inositol pentakisphosphate dephosphorylation by the phytate-degrading enzyme from Aspergillus niger. Substrate

Km (µmol L–1) / kcat (s–1)

InsP5 generated by the B. amyloliquefaciens ATCC 15841 enzyme InsP5 generated by the B. amyloliquefaciens 45 enzyme InsP5 generated by the B. subtilis 168 enzyme D-Ins(1,2,4,5,6)P5

162±10 / 155±11

132±10 / 139±9

Fig. 2. Suggested degradation pathways of phytate by phytatedegrading enzymes Ins(2,4,6)P3 was not available as a reference compound; therefore it could not be excluded as a degradation ), occurs only under extreme conditions (prointermediate. ( longed incubation times, high enzyme concentration); ( ), minor pathways of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzymes; ( ), major pathway of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzymes.

149±15 / 142±9 157±12 / 151±14

Note: B. amyloliquefaciens, Bacillus amyloliquefaciens; B. subtilis, Bacillus subtilis. The data are mean values ± standard deviation of five independent experiments. Values were not found to be significantly different (P < 0.05).

and tetrakisphosphate isomers generated by the phytatedegrading enzymes under investigation is well established. A clear identification of the formed myo-inositol trisphosphate isomers by HPIC has not been possible until now, since not all theoretically existing isomers were available. Ins(2,4,5,6)P4 may be degraded to D/L-Ins(2,4,5)P3, Ins(2,4,6)P3, and Ins(4,5,6)P3. According to HPIC, Ins(4,5,6)P3 has to be excluded as an intermediate, since this myo-inositol phosphate elutes well resolved from the InsP3 peaks generated during myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzymes under investigation (Fig. 1), but it is not possible to discriminate between the formation of D/L-Ins(2,4,5)P3 and Ins(2,4,6)P3 on the HPIC system, since pure Ins(2,4,6)P3 is not available as a reference compound. Ins(2)P may be produced from D-Ins(1,2,5,6)P4 via the six different routes indicated in Fig. 3. Thereby, D-Ins(1,2,5)P3, D-Ins(1,2,6)P3, and D-Ins(2,5,6)P3, as well as D-Ins(1,2)P2, D-Ins(2,5)P2, and D-Ins(2,6)P2 may occur. According to HPIC, D-Ins(1,2)P2 and D-Ins(2,5)P2 have to be excluded as intermediates, since these myo-inositol bisphosphates elute well resolved from the InsP2 peak generated during myoinositol hexakisphosphate dephosphorylation by the phytatedegrading enzymes under investigation (Fig. 1). In addition, only a myo-inositol trisphosphate intermediate co-eluting with D-Ins(1,2,6)P3 could serve as a precursor of Ins(2)P. Thus, Ins(2)P is generated from D-Ins(1,2,5,6)P4 via D-Ins(1,2,6)P3 and D-Ins(2,6)P2, whereby degradation of D-Ins(1,2,6)P3 is very slow and was therefore only observed in the presence of high enzyme concentration during prolonged incubation. The phytate-degrading enzymes under investigation also exhibited, to a small extent, 6-phytase activity. Thereby, D/LIns(1,2,3,4,5)P5 is generated, which may be linked to pathway 2 by dephosphorylation to D-Ins(1,2,5,6)P4. A further minor pathway seems to proceed from D-Ins(1,2,4,5,6)P5 via DIns(1,2,4,6)P4 to finally Ins(2,4,6)P3 or Ins(2)P via DIns(1,2,6)P3 and D-Ins(2,6)P2. When you compare the major hydrolysis pathways of enzymatic myo-inositol hexakisphosphate degradation by phytate-degrading enzymes from Bacillus spp. elucidated in this study with those reported by Kerovuo et al. (2000), the following differences are striking. Even under identical conditions for phytate degradation, two major myo-inositol

trisphosphate intermediates were found by HPIC analysis, whereas Kerovuo et al. (2000) have reported only one major myo-inositol trisphosphate peak using metal-dye detection (MDD)–HPLC analysis. Furthermore, the myo-inositol tetrakisphosphate isomer Ins(1,2,3,5)P4 reported to be an intermediate of phytate hydrolysis by a phytate-degrading enzyme from B. subtilis (Kerovuo et al. 2000) was not found to be one in this study; instead, D-Ins(1,2,5,6)P4 was identified as an intermediate. Because of the very high sequence identity of the enzymes, different degradation pathways are not very likely as the cause of the observed differences, especially if you take into account that all three enzymes included in this study exhibit almost identical properties in myo-inositol phosphate degradation. Incorrect correlation of the myo-inositol phosphate isomers and the elution volumes in the HPLC systems used may be another reason for the observed differences. However, since myo-inositol hexakisphosphate dephosphorylation by the phytatedegrading enzyme from wheat, deduced using NMRtechnique (Nakano et al. 2000), and the phytate-degrading enzyme D21 from spelt, an old wheat variety, using a com© 2002 NRC Canada



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bination of HPIC analysis and kinetic studies (Greiner and Larsson Alminger 2001), gave identical results, the suitability of the latter to reveal the stereospecificity of enzymatic myo-inositol hexakisphosphate degradation has already been demonstrated. Furthermore, it was suggested by Kerovuo et al. (2000) that the D/L-Ins(1,2,3,4,5)P5-intermediate generated by the phytatedegrading enzyme of B. subtilis remains tightly bound to the active site of the phytate-degrading enzyme until the phosphates both in 6- and 4-positions are removed, since the D/LIns(1,2,3,4,5)P5-intermediate was found in small quantity only. The resulting Ins(1,2,3,5)P4 is then released from the phytate-degrading enzyme from B. subtilis. Identification of the myo-inositol tetrakisphosphate isomers generated by dephosphorylation of D-Ins(1,2,4,5,6)P5, D-Ins(1,2,3,4,5)P5, and L-Ins(1,2,3,4,5)P5 (identical with D-Ins(1,2,3,5,6)P5) by the phytate-degrading enzymes under investigation confirmed that D-Ins(1,2,3,4,5)P5 and D-Ins(1,2,3,5,6)P5, could serve as the precursor to Ins(1,2,3,5)P4, but dephosphorylation of DIns(1,2,4,5,6)P5 results in the formation of Ins(2,4,5,6)P4 and D-Ins(1,2,5,6)P4. According to this data, none of the suggested degradation pathways could be excluded. In addition, it was proposed that myo-inositol phosphate substrates are bound to the active site of the enzymes by two adjacent phosphate residues on the myo-inositol ring, and one of these two phosphate residues is cleaved during the catalytic reaction. This suggestion was supported by kinetic evaluation of the hydrolysis of different myo-inositol phosphates and computer-modelled substrate-binding (Kerovuo et al. 2000; Shin et al. 2001). Our data is not in contradiction to this suggestion. When comparing myo-inositol phosphates with adjacent phosphate residues, myo-inositol phosphates without adjacent phosphate residues such as Ins(2)P showed a significantly lower affinity for the phytate-degrading enzyme studied, and their maximal rates of hydrolysis were also clearly lower. The kinetic parameters for the hydrolysis of Ins(2)P and phytate by the phytate-degrading enzyme studied are in good agreement with those reported for the phytate-degrading enzyme from B. amyloliquefaciens DS11 (Shin et al. 2001). The kinetic parameters for the degradation of the mixture of myo-inositol trisphosphate intermediates generated by the phytate-degrading enzymes studied are more comparable with the kinetic parameters determined for the hydrolysis of Ins(2)P and not with those determined for the hydrolysis of D-Ins(1,2,6)P3. Since this myo-inositol trisphosphate mixture consists of D-Ins(1,2,6)P3 and D/LIns(2,4,5)P3 or Ins(2,4,6)P3, and the latter are resistant to further degradation by the phytate-degrading enzymes under investigation, they may act as an inhibitor for the degradation of D-Ins(1,2,6)P3. This supports Ins(2,4,6)P3 as the final product of pathway 2; because of missing adjacent phosphate residues, it should be a poorer substrate for the phytate-degrading enzymes studied. Thus, pathway 2 would be identical to pathway 1 reported by Kerovuo et al. (2000) for the phytate-degrading enzyme from B. subtilis. An explanation for the further degradation of D-Ins(1,2,6)P3 could be the higher enzyme activity and longer incubation times in comparison with the conditions used by Kerovuo et al. (2000), especially since Powar and Jagannathan (1982) also identified myo-inositol monophosphate as the end prod-

Can. J. Microbiol. Vol. 48, 2002 Fig. 3. Theoretically possible degradation pathways from the myo-inositol tetrakisphosphate isomer D-Ins(1,2,5,6)P4 to Ins(2)P. ( ), to be excluded according to the results of HPIC (Fig. 1); ( ), to be excluded, since only a myo-inositol trisphosphate intermediate co-eluting with D-Ins(1,2,6)P3 could serve as a precursor of Ins(2)P; ( ), major pathway of D-Ins(1,2,5,6)P4 dephosphorylation by the phytate-degrading enzymes.

uct of phytate degradation by a phytate-degrading enzyme from B. subtilis. A further explanation would be the presence of a contaminating, nonspecific acid phosphatase in the enzyme preparation used in the studies, but such a contamination does not explain the difference in the myo-inositol tetrakis- and trisphosphate intermediates determined during phytate degradation by the phytate-degrading enzymes and B. subtilis (Kerovuo et al. 2000). According to computer-modelled substrate-binding (Shin et al. 2001), a superposition without steric clash is limited to the pairs of (C-3 and C-4) and (C-6 and C-1). This observation suggests that, initially, only the phosphate residues at C-3 and C-6 are accessible for enzymatic cleavage. Removal of C-3 allows superposition of (C-1 and C-2), whereas removal of C6 allows superposition of (C-4 and C-5). Thus, computermodelled substrate-binding confirms the phytate hydrolysis pathways suggested by Kerovuo et al. (2000). On the other hand it was shown that computer-modelled substrate binding is not always capable of describing the reality. For example, it was shown by biochemical analysis of hydrolysis intermediates that the phytate-degrading enzyme from E. coli preferentially hydrolyses the 6-phosphate of phytate (Greiner et al. 2000). However, only phytate modelled with the 3phosphate in the active site fully accounted for the observed electron density. Although the discrepancy in the electron density maps while binding other phosphate residues of phytate is clear, the overall structural difference is very small, and there is no obvious difference in terms of observed interaction. Hence, there is no apparent structural explanation as to why one orientation is preferred over the other (Lim et al. 2000). According to Kerovuo et al. (2000), the phytate-degrading enzyme of B. subtilis is the only one generating a myoinositol phosphate intermediate (D/L-Ins(1,2,3,4,5)P5), which remain tightly bound to the active site of the phytatedegrading enzyme until the phosphates in both the 6- and 4positions are removed. Furthermore, it is the only phytatedegrading enzyme which was reported to remove the axial phosphate residue at the C-2 position of the myo-inositol © 2002 NRC Canada



Greiner et al.

ring. According to our data, the phytate-degrading enzymes do not exhibit this unique feature. To establish the phytate degradation pathway of the phytatedegrading enzymes from Bacillus spp., further work has to be carried out. A promising approach is the separation and purification of the individual intermediates and their analysis by NMR.

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