Plant Physiol. (1994) 106: 1489-1495

Specificity of Hydrolysis of Phytic Acid by Alkaline Phytase from Lily Pollen

Laura Barrientos, Jonathan J. Scott, and Pushpalatha P. N. Murthy* Chemistry Department, Michigan Technological University, 1400 Townsend Drive, Houghton, Michigan 49931

(L.B., P.P.N.M.); and Department of Biology, Mount Union College, Alliance, Ohio 44601 (J.J.S.)

Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (I- 1,2,3,4,5,6-P,). A number of phytases with varying specificities, properties, and localizations hydrolyze phytic acid present in cells. l h e specificity of hydrolysis of phytic acid by alkaline phytase from lily (Lilium longiflorum 1.) pollen is described. Structures of the intermediate inositol phosphates and the final product were estab- lished by a variety of nuclear magnetic resonance techniques ('H-, 31P-, and 3'P-'H-detected multiple quantum coherence spec- troscopy, and total correlation spectroscopy). On the basis of the structures identified we have proposed a scheme of hydrolysis of phytic acid. Initial hydrolysis of the phosphate ester occurs at the D-5 position of phytic acid to yield the symmetrical I-1,2,3,4,6-P5. The two subsequent dephosphorylations occur adjacent to the D- 5 hydroxyl group to yield I-1,2,3-P3 as the final product. Alkaline phytase differs from other phytases in the specificity of hydrolysis of phosphate esters on the inositol ring, its high substrate specificity for phytic acid, and biochemical properties such as susceptibility to activation by calcium and inhibition by fluoride. l h e physiolog- ical significance of alkaline phytase and the biological role of I- 1,2,3-P3 remain to be identified.

Phytic acid, myo-inositol hexakisphosphate, is a major con- stituent of seeds and pollen grains (1-5% of dry weight) (Loewus, 1990; Raboy, 1990). In mature lily (Lilium longiflo- rum L.) pollen and seeds, phytic acid is localized in mem- brane-bound phytic-rich granules. The presence of phytic acid in plant cells has been known for some time (Loewus, 1990; Raboy, 1990). However, it was only recently recognized that phytic acid is present in virtually all mammalian cells in concentrations higher than most other inositol phosphates (Menniti et al., 1993) and may function as a neurotransmitter (Vallejo et al., 1988). The discovery that I-1,4,5-P3 plays a crucial role in calcium cellular signaling (Bemdge et al., 1989) has greatly increased interest in the structure, metabolism, and biological role of inositol phosphates, including phytic acid.

The primary enzymes responsible for the degradation of phytic acid are phytases. Phytases are a special class of phosphatases that catalyze the sequential hydrolysis of phytic acid to inositol phosphates and, in some cases, to inositol. Phytases occur in a variety of organisms including plants, fungi, and animals (reviewed by Cosgrove, 1980a). A variety of phytases differing in pH optima, substrate specificity, and

* Corresponding author; fax 1-906-487-2061.

specificity of hydrolysis have been identified in plants and fungi (Cosgrove, 1980a, 1980b). Acid phytases from wheat bran and Aspergilli have been extensively studied and the stereospecificity of hydrolysis has been well established (Cos- grove, 1980b). Based on the specificity of initial hydrolysis, two classes of acid phytases are recognized by the Intema- tional Union of Pure and Applied Chemistry and the Inter- national Union of Biochemistry (IUPAC-IUB, 1975), the 6- phytase, found in plants, and the 3-phytase, found in fungi. The 6-phytase hydrolyzes the phosphate ester at the L-6 (or D-4) position of phytic acid, and the 3-phytase hydrolyzes the phosphate ester at the D-3 position.

Recently, investigators in the laboratory of F.A. Loewus (Scott and Loewus, 1986) isolated a new form of phytase from lily pollen that exhibits some unusual characteristics. It has a pH optimum of 8, it is not inhibited by fluoride at concentrations that inhibit acidic phytase activity by 90% (10 m), and it is activated by calcium ions (Baldi et al., 1988). Also, alkaline phytase removes only three phosphates from IP6 to yield IPS as the final product (Baldi et al., 1988; Loewus et al., 1990). More recently the presence of alkaline phytase in a variety of seeds was demonstrated (Scott, 1991).

In this paper we describe our research effort to determine the specificity of hydrolysis of phytic acid by alkaline phytase from lily pollen. Previous investigations of phytases have relied heavily on indirect methods such as degradation of inositol phosphates to acyclic sugars by a sequence of reac- tions (periodate cleavage, reduction, and dephosphorylation) and subsequent identification of the final product by a variety of chromatographic techniques (Lim et al., 1973; Cosgrove, 1980b). In this investigation, structures of the intermediate and final inositol phosphates were established by direct struc- tural analysis (1D- and 2D-NMR techniques). The enzyme exhibited markedly different catalytic characteristics both in the specificity of hydrolysis and in the final product generated.

Abbreviations: lD, one-dimensional; 2D, two-dimensional; HMQC, 'H-detected heteronuclear multiple quantum coherence; HOHAHA, homonuclear Hartmann-Hann; HVE, high-voltage paper electrophoresis; IPS, IF5, Ir4, IF,, IF2, and IF1, myo-inositol hexakis-, pentakis-, tetrakis-, tris-, bis-, and mono-phosphates, respectively, with numbering of the D configuration as appropriate; Jax.ax, Jax.q,

Jq.q, spin-spin coupling constants between vicinal diaxial, axial- equatorial, and diequatorial protons, respectively; TOCSY, total cor- relation spectroscopy. _.

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1490 Barrientos et al. Plant Physiol. Vol. 106, 1994

MATERIALS AND METHODS

Preparation of Alkaline Phytase

Preparation of alkaline phytase followed the method of Baldi et al. (1988). Lilium longiflorum L. cv Nellie White (1991 harvest) was kindly supplied by F.A. Loewus of the Institute of Biological Chemistry at Washington State University. All enzyme preparation procedures were carried out on an ice bath or in a cold room at 4OC. Ten grams of pollen was suspended in 50 mL of 0.01 M Tris-HCI, pH 7.0, containing 0.5 m~ GSH. The suspension was stirred with a glass rod until most of the pollen kit had adhered to the rod and could be easily removed. An additional 50 mL of the same buffer containing 1% cetylpyridinium bromide was added to the pollen suspension to provide a final concentration of 0.5% detergent. The suspension was homogenized in a motorized glass-glass homogenizer with the aid of a small amount of clean sand. Homogenization proceeded until microscopic ex- amination revealed that most of the pollen grains had been broken. This was generally accomplished after 20 up-and- down strokes with the motorized homogenizer. The homog- enate was centrifuged at 20,OOOg for 20 min and the super- natant was filtered through Whatman No. 4 filter paper. The filtered solution was dialyzed against 0.01 M Tris-HC1 buffer (pH 7.0) containing 0.5 m~ GSH at 4OC. In some experiments the filtered extract was used without further purification. In other cases the alkaline phytase was further purified by CaC12 precipitation and ion-exchange chromatography as described below.

The filtrate was brought to 0.01 M CaCI2 by addition of an appropriate amount of 1 M CaC12 and then stirred for 15 min. The resulting precipitate was removed by centrifugation as described above and the supernatant, containing most of the alkaline phytase, was applied to a column (26-mL volume) of DEAE Sepharose CL-6B that had been equilibrated with 0.01 M Tris-HC1, pH 7.0, containing 0.05 m~ GSH. The column was washed with 100 mL of buffer followed by a linear gradient of O to 0.6 M KCl in the buffer. Most of the alkaline phytase eluted between 0.02 and 0.17 M KC1.

Both crude, filtered extracts and DEAE-purified alkaline phytase preparations were dialyzed, concentrated by ultrafil- tration, and stored at 4OC. Specific activity of alkaline phytase preparations varied from 0.1 nkat/mg protein in crude, fil- tered extracts to 0.32 nkat/mg protein in DEAE-purified preparations. In the presence of sodium fluoride (20 m), an inhibitor of acid phytase and acid phosphatase, HVE and NMR spectroscopy indicated that both crude, filtered extracts and DEAE-purified samples gave rise to the same interme- diates and products. The alkaline phytase proved to be un- stable and large losses of activity occurred during the purifi- cation procedures.

Phytase Assays

The reaction mixture for the phytase assay contained 0.1 M Tris-HC1 (pH 8.0), 1 m~ sodium phytate, 1 m~ CaC12, and an aliquot (usually 0.1 mL containing protein) of phytase sample in a total volume of 1.2 mL. After incubation at 37OC for 60 min, the reaction was stopped by addition of 0.8 mL of 10% TCA. The precipitated protein was removed by

centrifugation and the supernatant was analyzed for Pi (Ames, 1966). Protein concentration was determined by the Coomassie blue dye-binding method (Bradford, 15176).

HVE Analysis of Inositol Phosphates

To analyze the inositol phosphates released by alkaline phytase treatment of phytic acid, a solution of (1.2 M Tris- HCl, pH 8.0, 2 m~ CaC12, and 20 m~ sodium fluoride was mixed with an equal volume of phytase preparation. Sodium fluoride was included in the reaction mixture to inhibit acid phytase activity, which was present in the alkaline phytase preparations. The reaction mixture was allowed to incubate on a water bath at 37OC. Aliquots were removed at timed intervals and spotted onto Whatman No. 1 chromatography paper, and the inositol phosphates were separated by HVE in a Gilson model D electrophorator (Agranoff et al., 1983). Electrophoresis was carried out at 4 kV for 15 miri in 0.06 M sodium oxalate buffer (pH 1.54). After electrophoresis, the dried paper was sprayed with Clark/Dawson spray reagent (Clark et al., 1981) to locate the inositol phosphate:,. R F values of inositol phosphates formed in the reaction and those of other inositol phosphates are as follows: Ir6, 1.0; 1-1,2,3,5,6-

0.81; I-1,2,3-P3, 0.72; I-1,2,6-P3, 0.68. Preparative HVE of inositol phosphates was performed by streaking 0.4-mL sam- ples of reaction mixture along a 16-cm line on Whatman No. 1 chromatography paper followed by electrophoresis as de- scribed above. Inositol phosphate-containing arcas of the paper were cut out and washed with absolute ethanol to remove oxalic acid and Varsol oil remaining from electropho- resis. The paper was air dried and then extracted twice with distilled water to elute the inositol phosphates. The washes were combined, passed through a 0.45-pm filter, and lyoph- ilized prior to NMR analysis.

P5, 0.92; 1-1,2,3,4,6-P5, 0.88; I-1,2,5,6-P4, 0.86; I-1,2,3,6-P4,

NMR Spectroscopy

Spectra were recorded on a 500-MHz Varian VXR-500 spectrometer. Dried samples (0.5-2.0 mg) were dssolved in D20 (0.7 mL) and pH adjustments were made with perdeu- terated acetic acid. All spectra were recorded at 25OC. 1-D 'H NMR spectra were obtained at 499.84 MHz. '€I chemical shifts were referenced to the residual proton absorption of the solvent, D20 (6 4.67). The acquisition conditions were as follows: spectral windows, 6738 Hz; pulse width, 50 to 90° tipping angle. Typically, 64 scans with a recycle delay of 6 s between acquisitions was collected. The residual H 2 0 reso- nance was suppressed by a 2-s selective presatura tion pulse.

Proton-coupled or decoupled 31P-NMR spectra were ob- tained at 203.33 MHz. 31P chemical shifts were referenced to extemal phosphoric acid (85%). The acquisition conditions were as follows: spectral window, 10,000 Hz; pulse width, 15 ps; delay between pulses, 3 s.

31P-HMQC was obtained using an inverse detecíion probe. The pulse sequence was that of Summers et d. (1986). Experimental details and processing parameters are given in the figure legends.

TOCSY, also called HOHAHA, data sets went obtained with a 'H probe using the pulse sequence of Gries lnger et al.

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Specificity of Hydrolysis of Phytic Acid by Alkaline Phytase 1491

(1988). Experimental details and processing parameters aregiven in the figure legends.

RESULTS

Products of alkaline phytase-catalyzed phytic acid hydrol-ysis were separated by HVE. Comparison of migratory be-havior of the inositol phosphates formed with referencecompounds showed the formation of IP5, IP4, and IP3. Thesuccessive appearance of IP5, IP4, and IP3 with time (Fig. 1)suggests sequential hydrolysis of phosphate esters of phyticacid, which is consistent with observations of acid phytasesuch as wheat bran phytase-catalyzed reactions (Lim et al.,1973; Cosgrove, 1980). However, unlike the products formedby wheat bran phytase, only one isomer of IP5, IP4, and IP3was observed by HVE under conditions that clearly separatedall isomers of IP5, IP4, and IP3 that were in our possession(RF values are presented in "Materials and Methods"). Also,as seen in Figure 1, the final product of the reaction is IP3.Consistent with previous observations, further hydrolysis ofIP3 was not detected for an additional 48 h (Baldi, 1988).

To isolate IP5, IP4, and IP3 produced by alkaline phytase,an aliquot of the reaction mixture was frozen at differenttimes, the products were separated by HVE, and the inositolphosphates were extracted from the paper. To establish thestructure of the inositol phosphates as well as to establish thepurity of the compounds, the inositol phosphates were ana-lyzed by 1D('H and 31P)- and 2D-NMR spectroscopy. NMRinvestigation was based on the premise that dephosphoryla-tion of phytic acid would affect the NMR spectroscopiccharacteristics of phytic acid in a diagnostic manner, (a)Dephosphorylation would lead to upfield shift of the aproton by approximately 0.5 to 1.0 ppm (Cerdan et al., 1986).

,IP6

•IPs,IP4

,IP3

6 A.A 4.2 4.0 3.8 3.6 3.4 3.2 Don

E

, Origin

Figure 2. 'H-NMR spectra of IP6 and inositol phosphates derivedfrom the action of alkaline phytase on IP6. Reaction was stopped atdifferent times by freezing. The inositol phosphates were separatedby HVE, extracted from the paper, and lyophilized. A, IP6 at pH12.0. B, IP5 at pH 5.0. C, Mixture of IP4 and IP3 at pH 5.2. D, IP4 atpH 5.0. E, IP3 at pH 5.0. Exponential line broadening of 0.5 Hz wasapplied. Digital resolution is 0.2 Hz/point. The singlet, labeled S, at& 3.58 in C and E is due to contamination from a new batch of filterpaper used during HVE. See also legend to Figure 4.

Figure 1. HVE separation of inositol phosphates produced by al-kaline phytase-catalyzed hydrolysis of phytic acid. Aliquots of thereaction mixture were removed at the times indicated and imme-diately frozen. The reaction mixtures were separated by HVE andsprayed with molybdate spray reagent to visualize the phosphates. www.plantphysiol.orgon January 21, 2020 - Published by Downloaded from

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1492 Barrientos et al. Plant Physiol. Vol. 106, 1994

Figure 3. Two-dimensional 3'P-decoupled HMQC of IP, with attached 'H and proton- decoupled 31P spectra. IPS was obtained as described in Figure 2. The pH was 5.0. The sweep width was 410 Hz in t h e F1 dimension and 2099 Hz in the Fz dimension. A total of 128 t, increments, each consisting of 24 tran- sients with relaxation delay of 2.5 s between successive transients, were obtained. A mixed Lorentzian and Ciaan window was applied in the F2 dimension and a shifted Cian window was applied in the F1 dimension. The data matrix was expanded to a 51 2 x 128 real matrix, resulting in digital resolution of 3.2 Hz/point in the F1 and 2.1 Hz/point in t h e Fz dimensions.

(b) Dephosphorylation would significantly affect the splitting pattem of the inositol ring proton because of loss of the proton-phosphorous coupling. The splitting pattem of pro- tons on the inositol ring is due to coupling with two vicinal protons, one on either side (Jax-eq about 2-3 Hz, Jax.ax about 8-10 Hz, and Jq-eq about 2-3 Hz) and with the phosphorous (JH-P about 8-10 Hz); therefore, the presence or absence of H-P coupling would-be obvious. (c) The presence of a plane of symmetry in the inositol phosphates formed would be indicated by the number of chemically distinct sets of reso- nances. Four distinct sets of resonances would result if there were a plane of symmetry and six would result if there were none. (d) 31P-HMQC would provide information on H-P connectivities and thereby provide additional proof of struc- tural assignment. (e) Further proof of structures could be obtained by specific and broad-band homonuclear and het- eronuclear decoupling experiments.

The 'H-NMR spectra of IP6 and the hydrolytic products IPS, Irr, and IP3 isolated from alkaline phytase-catalyzed reaction mixture are compared in Figure 2. The spectrum of IP, (Fig. 2A) consists of three sets of protons in the ratio 1:2:3. Consistent with observations that equatorial proton reso- nances are generally downfield of axial protons, the one equatorial proton H(2), at 6 4.8, is split into a triplet of doublets, which appears as broad doublets because of one large JH(Z)+ coupling and two small Jax.eq vicinal coupling to H(l) and H(3). The enantiotopic H(4) and H(6) protons at 6 4.35 are split into quartets due to two J,,.,, vicinal couplings with protons on either side and one JH-P coupling, all of similar magnitude. The enantiotopic H(1) and H(3) protons overlap with the H(5) proton at approximately 6 4.08. The H(5) proton is split into a quartet because of two vicinal Jax.ax

coupling and one JH-P coupling, all of similar magnitude. The H(1) and H(3) protons are split into broad triplets because of one J.x.ax coupling and a JH-P coupling of similar magnitude and a small Jax.q coupling with the H(2) proton.

H (2) L A - I

1.0

1.2

1 . 4

1 .6

4 . 0

1 3

-7 6 4 4 4 2 4 0 3 8 3 6 3 4 3 2

F2 lppm)

IPS (Fig. 2B) shows four sets of resonances in the intensity ratio 1:2:2:1, indicating a plane of symmetry in the molecule. The presence of a plane of symmetry suggests that the free hydroxy group must be at the C(2) or C(5) position. The most noticeable changes compared to IPS are the upficrld shift of the H(5) proton by about 0.5 ppm, the change in splitting pattem from a quartet to a sharp triplet, and the change in the reduction in intensity of the multiplet at abou- 6 4.1. The presence of the broadened doublet due to H(;!) at 6 4.8 suggests that Ir5 is I-1,2,3,4,6-P5. Consistent with the assign- ment, 31P-HMQC (Fig. 3) shows H-P connectivities at the H(2), H(l), H(3), H(4), and H(6) protons and none for the H(5) proton. The 31P resonances in the F1 dimension in the ratio 22: 1 again indicate a plane of symmetry in th. molecule.

When a mixture of IP4 and IP3 was analyzcmd, a more complicated proton spectrum (Fig. 2C) was obtained. To separate resonances due to IP4 from those dues to IP3, a TOCSY experiment was performed (Griesinger, 1988; Nak- anishi, 1990). The TOCSY technique, very similar to the HOHAHA technique, is useful for determining a network of mutually coupled protons, because in a TOCSY experiment multiple-bond coherence transfer can take place (Nakanishi, 1990). Since all protons on an inositol ring arc part of a connected spin system, all protons on IP, should show con- nectivity either due to direct coupling or due to long-range magnetization transfer. The 2D spectrum (Fig. 4) shows two networks of spin systems, one giving rise to four peaks as indicated by arrow A, and another giving rise to six peaks as indicated by arrow B. The presence of six sets of resonances in spin system B indicates a lack of symmetry in the molecule. The most noticeable change in spin system B cclmpared to IPS is the upfield shift of only one resonance, the H(4) [or enantiotopic H(6)] resonance, by about 0.5 ppin and the change in splitting pattem from a quartet to a hiplet, indi- cating loss of a coupling partner. Resonances closc? to 6 4.8, 6 4.3, and 6 4.1 have not changed appreciably in chunical shift

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Specificity of Hydrolysis of Phytic Acid by Alkaline Phytase 1493

I

't F 1 ' K *

5 1,3 4,6

4 . 6 4 . 4 4 . 0 3 . 6 3 . 2

F2 (ppm)

Figure 4. Two-dimensional TOCSY spectra of a mixture of lP4 and IP, with attached 'H spectra. The pH was 5.2. The sweep width employed in both the F1 and Fz dimensions was 2200 Hz. A total of 512 tl increments, each consisting of 24 transients with relaxation delay of 2 s between successive transients, were obtained. A sinebell window was applied in both dimensions and the data matrix was expanded to a 512 X 512 real matrix. Digital resolution was 4.3 Hz/point in both dimensions. The singlet at 6 3.65, labeled S, is due to contamination from a new batch of filter paper used during HVE.

or splitting pattem, indicating phosphorus couplings. There- fore, spin system B must be the tetraphosphate. H(5) of Ir4 shows direct coupling to H(4) and H(6) and long-range connectivities to H(l), H(3), and H(2). Additional confirma- tion of these structures was obtained by purifying IP4 and analyzing the NMR spectra (Fig. 2D). The spectrum in Figure 2D is very similar to spin system B (Fig. 4). Enantiotopic protons cannot be distinguished by NMR, so IP4 could be the structure I-1,2,3,6-P4 shown in Figure 2D or its enantiomer I-1,2,3,4-P4. Further confirmation of this assignment of phos- phorylated positions is provided by 31P-HMQC (Fig. 5), which shows H-P connectivities at H(1), H(2), H(3), and H(6) or its enantiotopic H(4).

The presence of four sets of resonances in spin system A (Fig. 4) suggests a plane of symmetry in the molecule. The upfield shift of H(6) [or enantiotopic H(4)] compared to IP4 and the change of quartet to triplet are consistent with removal of phosphate from H(6) [or H(4)]. The chemical shifts of H(2), H(l), H(3), and H(4) show relatively small

changes compared to Ir4, and the splitting pattems of H(l), H(2), and H(3) show the presence of H-P coupling. H(5) of Ir3 shows direct coupling to H(4) and H(6) and also clearly shows connectivities to H(1), H(3), and H(2). Additional confirmation of this structure was obtained by purifying IP3 and analyzing the NMR spectrum (Fig. 2E), which is very similar to that suggested by spin system A in Figure 4. The NMR spectra in Figure 4 and Figure 2D are consistent with the structure I-1,2,3-P3.

DISCUSSION

A complete understanding of the biological role of phytic acid and inositol phosphates requires detailed knowledge about the reaction specificity and biochemical characteristics of the enzymes involved in its metabolism. Phytases are the primary enzymes that catalyze the hydrolysis of phytic acid. In this study the specificity of hydrolysis of phytic acid by alkaline phytase was established. Establishing the specificity

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1494

Figure 5. Two-dimensional "P-decoupled HMQC of lP4 with attached 'H and proton- decoupled "P spectra. IP4 was obtained as described in Figure 2. The pH was 5.5. The sweep width was 2458 Hz in the F, dimension and 859 Hz in the F2 dimension. A total of 128 t1 increments, each consisting of 32 transients with relaxation delay of 2.5 s between succes- sive transients, were obtained. A shifted Cian window was applied in both dimensions fol- lowed by expansion to a 128 x 256 real matrix. The resulting digital resolution was 9.6 Hz/point in t h e F1 dimension and 5.4 Hz/point in the F2 dimension.

Barrientos et al. Plant Physiol. Vol. 106, 1994

of hydrolysis of phytases has always been a challenge be- cause of the need to identify the structures of individual inositol phosphates in a mixture containing regio- and ster- eoisomers. In this investigation, the structures of the inter- mediates and final products were determined by analytical methods that would provide direct structural information. The use of a variety of 1D- and 2D- (TOCSY, 31P-HMQC) NMR techniques allowed us to establish unambiguously the structures of IPS and Ir3. NMR evidence suggests that the Ir4 formed is due to hydrolysis of phosphate adjacent to the D- 5 position. However, since NMR cannot distinguish between enantiotopic protons, at this time we cannot suggest which enantiomer of Ir4, I-1,2,3,4-P4, or I-1,2,3,6-P4, is produced. TOCSY is commonly used to determine networks of mutually coupled protons in macromolecules (Griesinger et al., 1988; Nakanishi, 1990). In this investigation, the TOCSY experi- ment was successfully used for a different purpose, to provide information that allows us to establish structures of individual compounds in mixtures, without resorting to separation. Our

I

v 2.6

5.0 4 . 8 4 . 6 4 . 4 4 . 2 4 . 0 3.8 3.6 3 . 4

F2 (ppm)

results point out the tremendous potential of TOCSY exper- iments for this purpose.

On the basis of the structures identified, we propose that the sequence of dephosphorylation of phytic acid by alkaline phytase is as shown in Figure 6. Alkaline phytase from lily pollen hydrolyzed the phosphate at the D-5 position of phytic acid to yield a symmetrical IPS. Further hydrolysis occurs adjacent to the free hydroxyl group. The two subsequent dephosphorylations occur on either side of the 5-hydroxyl group to yield I-1,2,3-P3, a symmetrical molecule, as the final product. Unpublished information had suggested that alka- line phytase from Typha latifolia also produces I-1,2,3-P3 (Loewus et al., 1990). Consistent with characteristics of other phytases (Cosgrove, 1980b), hydrolysis occurs adjacent to the free hydroxyl group and, therefore, the initial position of hydrolysis is a major determinant of subsequent points of attack. Attack adjacent to a free hydroxyl group could be due to greater nucleophilicity of the hydroxyl oxygen compared to the ester bond oxygen (Cosgrove, 1980b). The rates of

(I-l,2,3,4,5,6-P6) (1-1,2,3,4,6-P5) (1- 1 ,2,3.4-P4)

(I- 1,2,3,6-P,)

Figure 6. Alkaline phytase-catalyzed dephosphorylation of phytic acid.

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Specificity of Hydrolysis of Phytic Acid by Alkaline Phytase 1495

removal of the second and third phosphates are significantly lower, possibly due to reduced specificity for I r 5 and IP4 (Baldi, 1988) and inhibition by the phosphate released. Al- temative routes of hydrolysis of the intermediates leading to formation of multiple isomers of inositol phosphates were not observed. Formation of I-1,2,3-P3 as one of three inositol trisphosphate intermediates, which is subsequently hydro- lyzed to 1,2- or 2,3-IP2 and finally to PI, has been observed with wheat bran phytase (Lim et al., 1973; Cosgrove, 1980b).

Phytases that differ in substrate specificity, position of hydrolysis on the inositol ring, final product produced, and biochemical characteristics such as pH optima and suscepti- bility to inhibitors and activators have been found, sometimes in the same tissue (Cosgrove, 1980a, 1980b; Baldi et al., 1988). For example, unlike alkaline phytase, most acid phy- tases studied hydrolyze phytic acid a t the D-3 or L-6 (or D-4) positions. Also, acid phytases are able to catalyze complete dephosphorylation of phytic acid to inositol, whereas alkaline phytase is unable to catalyze dephosphorylation of Ir3. In addition, unlike alkaline phytase, altemative pathways of hydrolysis of IP5 to yield multiple isomers of I r4 , Ir3, and IP2 are observed with acid phytases such as wheat bran phytase (Lm et al., 1973).

The narrow substrate specificity of alkaline phytase along with other characteristics such as higher specificity for phytic acid, more stringent specificity of hydrolysis leading to un- common isomers of inositol phosphates, such as I-1,2,3-P3, and dependence on calcium ions for activation all raise ques- tions regarding the potential physiological role, such as a regulatory role, for this enzyme. In vivo, phytic acid can be hydrolyzed by a number of phytases. The biological signifi- cance of the numerous phytases and the intracellular roles of the products produced are unknown. In addition to I-1,4,5- P3, biological functions for a number of inositol phosphates such as I-1,3,4,5-P4 (Bemdge et al., 1989) and IP6 (Vallejo et al., 1988) have been suggested. It is likely that other inositol phosphates may have messenger functions. I-1,2,3-P3 joins the growing list of inositol phosphates whose physiological roles remain to be established.

ACKNOWLEDGMENTS

We would like to thank Prof. F.A. Loewus (Institute of Biological Chemistry, Washington State University, Pullman, WA) for his gen- erous gift of pollen grains and for many helpful discussions. We would like to gratefully acknowledge the generous and skillful as- sistance of Dr. Le, Mr. Johnson, and Dr. Jackson of the Max T. Rogers NMR Facility of Michigan State University for the 500-MHz NMR spectra. The NMR data were obtained on instrumentation that was purchased in part with funds from National Institutes of Health grant No. 1-S10-RRO4750, National Science Foundation grant No. CHE- 88-00770, and National Science Foundation grant No. CHE-92- 13241. Dr. Jonathan Scott would like to acknowledge Mount Union College for a Sponsored Travel and Research Grant that made this collaboration possible.

Received April 18, 1994; accepted August 8, 1994. Copyright Clearance Center: 0032-0889/94/106/1489/07.

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