Kinetic characterization of O-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases

Article abstract of DOI:10.1021/jf800597f

Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH. The K _m for phosphotyrosine ranged from 465 to 590 μM as opposed to 135 to 160 μM for phytate. The V_max, however, is 2-4 times higher for phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on the same order as it is for phytate (3.5 × 10 ⁶ to 1.6 × 10⁷ M^-1 s^-1); the pH versus activity profile for phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5°C higher to 70°C when phosphotyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity has been reported for the subgroup of HAP known as phytase.

Full text of DOI:10.1021/jf800597f

J. Agric. Food Chem. 2008, 56, 7467–7471 7467
Kinetic Characterization of O-Phospho-L-tyrosine
Phosphohydrolase Activity of Two Fungal Phytases
ABUL H. J. ULLAH,* KANDAN SETHUMADHAVAN, AND EDWARD J. MULLANEY
Commodity Utilization Research Unit, Southern Regional Research Center, ARS, United States
Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124
Fungal phytases belonging to “histidine acid phosphatase” or HAP class of phosphohydrolases that
catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called
phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5
were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester
bond of phosphotyrosine efficiently at acidic pH. The Km for phosphotyrosine ranged from 465 to 590
µM as opposed to 135 to 160 µM for phytate. The Vmax, however, is 2-4 times higher for
phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on
the same order as it is for phytate (3.5 × 106 to 1.6 × 107 M-1 s-1); the pH versus activity profile for
phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5
°C higher to 70 °C when phosphotyrosine was used as the substrate. Taken together, the kinetic
data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester
bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity
has been reported for the subgroup of HAP known as phytase.
KEYWORDS: Phytase; PTPase; phosphohydrolase; phosphomonoesterase; histidine acid phosphatase
INTRODUCTION
rylated amino acids with difficulty (data not presented), PhyB
phytase could degrade phosphotyrosine facilely. The other
two phosphorylated amino acids were hydrolyzed albeit slowly
by PhyB phytase (A. Ullah, unpublished observation). A non-
HAP phytase has been reported in Selenomonas ruminantium
that share the active site motif and overall three-dimensional
structure with protein tyrosine phosphatase (13). In this com-
munication we present kinetic data demonstrating that PhyB
phytases catalyze the dephosphorylation of phosphotyrosine. In
near anaerobic condition in the rumen, a unique phosphatase
with cysteine at the active site is implicated as being the
phosphoprotein phosphatase or PTPase (13). This is however
the first time that anyone is reporting PTPase activity in HAP
type of acid phosphatase. All the work described here was done
using two members of PhyB, namely, pH 2.5 optimum phytase
produced by A. niger (14) and A. awamori (12).
Under limited phosphate Aspergillus niger (ficuum) NRRL
3135 produces three acid phosphatases (1–3). Of the three, two
belong to subgroup “histidine acid phosphatase” or HAP; these
acid phosphatases are capable of cleaving phosphates from
phytic acid and, as such, are called phytases (4). The third
acid phosphatase, pH 6.0-optimum phosphomonoesterase, lacks
phytate degrading ability and belonged to “purple acid phos-
phatase” subclass of phosphomonoesterase (5). The phytase
(phyA) gene from A. niger NRRL 3135 was cloned, overex-
pressed, and commercialized as a feed additive for poultry and
swine industries (6, 7). The mechanism of hydrolysis of the
monoester bond by phytase was elucidated, the enzyme’s three-
dimensional structure was deduced, and the sensitive Arg, His,
and Asp residues were mapped (8, 9). The A. niger NRRL 3135
PhyB phytase, which cleaves phytate phosphates at pH 2.5 at
an accelerated rate than PhyA phytase, is also a well-studied
enzyme; its gene (phyB) was cloned and the three-dimensional
structure had been elucidated (10, 11). Both PhyA and PhyB
could hydrolyze synthetic substrate, p-nitrophenylphosphate, and
a variety of phosphorylated sugars (2). The phyB was also cloned
from Aspergillus awamori and overexpressed in mutant strains
of the same species of fungi (12). To ascertain whether fungal
PhyA and PhyB could hydrolyze phosphorylated amino acids,
we tested phosphotyrosine, phosphoserine, and phosphothreo-
nine as substrate. While PhyA could utilize all three phospho-
MATERIALS AND METHODS
Source of Phytase. A. niger (ficuum) NRRL 3135 was grown in
starch media supplemented with glucose, sodium nitrate, and other salts
(1). The culture media containing PhyB activity was harvested by
centrifugation at 12000 rpm in a Sorval GSA rotor in ice-cold
conditions. The enzyme, PhyB, was purified using sequential ion-
exchange chromatographies using MacroPrep S and Q columns (2).
The purified phytase had a specific activity of 3570 nKat/mg protein.
The Finase enzyme produced by A. awamori was originally obtained
from Alko Ltd. Biotechnology, SF-05200 Rajamaki, Finland, by Prof.
E. R. Miller of Michigan State University. Prof. Xin-Gen Lei of Cornell
University had subsequently received the same enzyme and made it
* To whom correspondence should be addressed. E-mail: abul.ullah@
ars.usda.gov.
10.1021/jf800597f This article not subject to U.S. Copyright. Published 2008 by the American Chemical Society
Published on Web 07/16/2008

7468 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Ullah et al.
available to us. The phytase was then purified by chromatographic
methods identical to the purification of A. niger PhyB as mentioned
above. The specific activity of this highly homogeneous preparation
was 4357 nKat/mg protein.
Phytase Assay. Phytase assays were carried out in 1.0 mL volume
at 58 °C in assay buffer, 25 mM glycine, pH 2.5, similar to the A.
niger phytase assay (1). The liberated inorganic ortho-phosphates were
quantitated spectrophotometrically using a freshly prepared AMA
reagent consisting of acetone, 10 mM ammonium molybdate, and 2.5
M sulfuric acid (2:1:1, v/v). Adding 2.0 mL AMA solution per assay
tube terminated phytase assay. After 30 s, 0.1 mL of 1.0 M citric acid
was added to each tube to fix the color generated by AMA reagent.
Absorbance was read at 355 nm after blanking the spectrophotometer
with appropriate control. Values were expressed as nkat/mL where kat
stands for katal (mole substrate converted per second). One international
unit (IU) is equivalent to 16.67 nkat.
Phosphotyrosine Phosphohydrolase Activity. The assay is identical
to phytase assay except that the substrate 10 mM phosphotyrosine was
used in incubations and the assay buffer was 25 mM glycine, pH 2.5
or 25 mM imidazole, pH 6.0; the final concentration of phosphotyrosine
being 750 µM. The enzyme concentration was held at 65 pmol for
PhyB and 112 pmol for Finase.
Phenylglyoxal Treatment. A 10 µL aliquot of the PhyB (650 pmol)
was mixed in 240 µL of phenylglyoxal (250 mM) in bicarbonate (125
mM, pH 8.3) buffer and 45 µL of Finase (1120 pmole) was mixed
with equal volume of phenylglyoxal (250 mM) in the same bicarbonate
buffer. Reaction with the enzyme was carried out at 37 °C for 30 min.
At the end of incubation, 2 µL of the sample was used for the
phosphotyrosine phosphohydrolase activity with one min incubation
time. The control experiments contained bicarbonate buffer without
phenylglyoxal.
Figure 1. The pH optima of the fungal phytases (PhyB) using phospho-
tyrosine as substrate. The buffers used were glycine (pH 1.0-3.0), acetate
(pH 3.5-5.5), and imidazole (pH 6.0-8.0).
myo-inositol Hexasulfate Treatment (MIHS). A 2 µL aliquot of
PhyB (130 pmol) and 10 µL of Finase (249 pmol) was pipetted into
assay tubes containing 25 mM glycine, pH 2.5 buffer raised to 58 °C.
A 50 µL aliquot of 10 mM MIHS was added before the addition of 10
mM phosphotyrosine. The phosphotyrosine phosphohydrolase activity
was measured after incubating for 1 min. Parallel control experiments
were run without MIHS.
RESULTS
The PhyB phytase, first identified as a pH 2.5 optimum acid
phosphatase, is capable of hydrolyzing the phosphomonoester
bond of phosphotyrosine. Based on protein sequence, Finase, a
recombinant phytase from A. awamori, is a homologue of A.
niger PhyB.
Phosphotyrosine Phosphohydrolase Activity as a Function
of pH. While performing the substrate selectivity study of A.
niger PhyB, we discovered that this phosphomonoesterase
(PhyB) could efficiently hydrolyze phosphotyrosine at pH 2.5,
which is the pH optimum of this class of phytase (unpublished
observation). Therefore, activity was measured as a function of
pH for A. niger PhyB and Finase using phosphotyrosine as the
substrate (15). The results are shown in Figure 1. Both the
enzymes show bihump pH optima, one at pH 2.5 and the other
at 6.0. The ratio of activity at pH 2.5 over pH 6.0 is much higher
for Finase (3:1) than it is for A. niger phytase (0.9:1).
Nonetheless, these two members of PhyB could hydrolyze
phosphotyrosine quite facilely.
Phosphotyrosine Phosphohydrolase Activity as a Function
of Temperature. The PhyB phytase from A. niger is a thermo-
tolerant biocatalyst performing optimally at 63 °C (2). To
determine the optimum temperature of phosphotyrosine phos-
phohydrolase activity catalytic measurements were done at
various temperatures. The results are shown in Figure 2. The
temperature optimum for A. awamori PhyB was found to be
70 °C, which is 7 °C higher than what it is for degrading
synthetic substrate, p-nitrophenyl phosphate (2). However, at
75 °C the activity of both phytases dropped precipitously.
Figure 2. Temperature optima of the fungal phytases using phosphoty-
rosine as substrate. Phytase assays were carried out at 58 °C in assay
buffer, 25 mM glycine, pH 2.5.
Phytate versus Phosphotyrosine Hydrolysis by PhyB.
Figure 3 shows the results of phytate and phosphotyrosine
hydrolysis catalyzed by fungal PhyB phytases. Phosphotyrosine
was hydrolyzed more efficiently than phytic acid. Figure 3A
shows the concentration dependent breakdown of both phytate
and phosphotyrosine by A. niger PhyB phytase while panel B
depicts the breakdown of phytate and phosphotyrosine by
recombinant A. awamori PhyB phytase (Finase). The A. niger
PhyB cleaved phosphate nearly 2.25 times faster from phos-
photyrosine than it did from phytate. The recombinant A.
awamori PhyB phytase demonstrated overall a higher rate of
phosphohydrolase activity, but was similar to A. niger PhyB in
its preferential hydrolysis of phosphotyrosine as compared to
phytate. In A. niger PhyB when phosphotyrosine was used as
substrate, the turnover number increased while the kinetic

Phytase Exhibiting PTPase Activity
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7469
Figure 3. Phosphohydrolase activity of fungal phytases as function of substrate concentration. Both the phytase and the phosphotyrosine phosphatase
assays were carried out at 58 °C in assay buffer, 25 mM glycine, pH 2.5.
Table 1. Kinetic Parameters and Catalytic Efficiency of PhyB Phytase and
and p-nitrophenylphosphate (p-NPP) consisted of a single peak
Finase for Phytate and Phosphotyrosine
with optimum pH at 2.5 (2). However, when phosphotyrosine
was used as the substrate, the PhyB phytases from both A. niger
and A. awamori gave two distinct pH optima, one at 2.5 and
the other at 6.0 (Figure 1). Based on the pH versus activity
profile, it seems as if phosphotyrosine binds to the active site
differently than phytate.
catalytic
Km
turnover
(number s-1
efficiency
(M-1 s-1
)
enzymes
substrate
phytate
phosphotyrosine
phytate
phosphotyrosine
(µM)
)
PhyB phytase
PhyB phytase
Finase
135
465
160
590
2178
4894
710
1.61 × 107
1.05 × 107
4.43 × 106
3.59 × 106
Earlier, it was noticed that PhyB had a higher temperature
optimum for synthetic substrate, p-NPP than PhyA phytase.
Previously, A. ficuum PhyB phytase was shown to be a
homodimer based on gel filtration in native state (2); it was
also shown to be more thermo-tolerant than PhyA phytase. Later,
the hydrodynamic radius (RH) of PhyB and Finase was found
to be 5.5 nm (180-kDa), which supports the dimeric state of
the native enzyme (A. Ullah, unpublished data). The dimeric
structure may play a role in enhancing the thermo-stability in
PhyB phytase. However, when phosphotyrosine was used as
the substrate, the enzyme was found to be even more thermo-
tolerant. The optimum temperature for phosphotyrosine hy-
drolysis was found to be 70 °C (Figure 2), which is 7 °C higher
than phytate degradation.
Finase
2118
efficiency decreased; similar result was observed when Finase
was used to hydrolyze phosphotyrosine (Table 1). Phytate shows
a higher affinity for binding to the active site as exemplified by
a lower Km. Like Finase, A niger PhyB phytase shows a higher
Km for phosphotyrosine (Table 1).
Phenylglyoxal Modification of PhyB. To examine if both
phytate and phosphotyrosine utilize the same active site in PhyB
subclass of phytase, both the proteins were treated with
phenylglyoxal to modify arginine in the active site. The results
are shown in Figure 4. Phenylglyoxal treated PhyB from both
A. niger and A. awamori were totally inactivated. Phosphoty-
rosine phosphohydrolase activity of the treated PhyB phytase
performed at both pH 3.0 and 6.0 were inactivated. This
indicates that both phytate and phosphotyrosine utilize the same
active site of phyB phytase.
myo-inositol Hexasulfate (MIHS) Inhibition of Phospho-
tyrosine Phosphohydrolase Activity. MIHS, both a substrate
analog and a known active site inhibitor of phytase (16) was
used to investigate whether the mechanistic enzymology of
PhyB subclass of phytase is same for phytate-driven and
phosphotyrosine-driven phosphohydrolase reaction. The result
of MIHS inhibition of phosphotyrosine phosphohydrolase
activity of both A. niger and A. awamori PhyB phytase is
shown in Figure 5. The enzymatic data clearly shows that
at pH 3.0 MIHS is a potent inhibitor of both the PhyB
phytases. However, at pH 6.0 MIHS had failed to inhibit the
phosphotyrosine phosphohydrolase activity. Therefore, pH
plays an important role in the three-dimensional structure of
the active site.
Phosphotyrosine was hydrolyzed more efficiently than
phytate by both the fungal PhyB phytases. The higher
turnover number for phosphotyrosine driven catalysis over
phytate reflects this enhanced catalysis. However, the affinity
for phytate was much stronger than phosphotyrosine (Table
1). The combined kinetic parameters of Km and turnover
number when taken together explain why kinetic efficiency
is not different in the case of these two PhyB enzymes for
phytate and phosphotyrosine.
A sensitive Arg residue in the active site of phytase was
implicated for catalyzing the phosphomonoester bond in phytate
(9). To test whether the same Arg is involved in the catalysis
of phosphotyrosine, both the PhyBs were treated with phenylg-
lyoxal, a known modifier of Arg. The modified PhyBs from
both the sources failed to hydrolyze phosphotyrosine at pH 3.0
and 6.0 indicating that the same active site for phytate hydrolysis
is used to cleave phosphotyrosine.
MIHS, a phytate analog, was developed as an active site
inhibitor for HAP subclass of acid phosphatase (15). At pH 3.0
the inhibitor bound to the substrate-binding domain of the active
site of both the PhyBs and did not allow hydrolysis of
phosphotyrosine; however, at pH 6.0 the catalysis of phospho-
tyrosine was not inhibited by MIHS (Figure 5). The precise
DISCUSSION
PhyB phytase from A. niger and A. awamori hydrolyzed
phosphotyrosine efficiently. The pH optimum for both phytate

7470 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Ullah et al.
Figure 4. Inhibition of phosphotyrosine phosphohydrolase activity of fungal phytases by phenylglyoxal. After inactivation the enzyme activity was performed
at 58 °C in assay buffer, 25 mM glycine, pH 2.5.
Figure 5. Inhibition of phosphotyrosine phosphohydrolase activity of the fungal phytases by MIHS. The MIHS treated enzyme was assayed for activity
at 58 °C in assay buffer, 25 mM glycine, pH 2.5.
molecular explanation of this pH dependency for MIHS to block
phosphohydrolase activity is lacking at this time; however, one
possible explanation for no inhibition at pH 6.0 might be due
to absence of negatively charged sulfate groups on MIHS at
pH 6.0. Perhaps the X-ray deduced three-dimensional structure
of PhyB in the presence of MIHS at pH 3.0 and 6.0 might
unravel this differential effect of pH on catalysis in the presence
of MIHS.
Protein-tyrosine-phosphatase or phosphotyrosine phos-
phatase (PTPase) are enzymes that catalyze the removal of
a phosphate group attached to a tyrosine residue. These
enzymes are very important in the control of cell growth,
proliferation, differentiation and transformation (17). Multiple
forms of PTPase have been characterized and could be
classified into two categories: soluble PTPases and trans-
membrane receptor proteins that contain PTPase domain(s).
One member of the PTP subclass, cysteine phosphatase, is
known to be able to degrade phytic acid (18). The active
site of cysteine phosphatase contains a conserved octa-peptide
with sequence HCXXGXXR and shows no sequence homol-
ogy with HAP subclass of acid phosphatase (E.C. 3.1.3.2
and E.C. 3.1.3.8) whose active site sequence is RHGXRXP.
Furthermore, PTPase or tyrosine phosphatases have a sig-
nature sequence in their active site, with a cysteine residue
that is essential for activity. The cysteine serves as the active
site nucleophile (19). However, in fungal HAP, 4-5 disulfide
bridges are formed by 8-10 Cys residues. Therefore, from
the perspectives of active site and sequence homology, these
two classes of phosphatases are markedly different. This is
the first documentation that PhyB from Aspergillus is capable
of efficiently hydrolyzing phosphotyrosine. It is noteworthy
that a new class of phytase related to PTPase was isolated
from several anaerobic bacteria from the rumen of cattle (19).
The observed diversity of this new class of phytase in the
rumen may account for the ability of ruminants to hydrolyze
phytate. Despite no homology between the active site of
phytase and PTPase both classes of enzymes degrade a variety
of phosphorylated metabolites such as myo-inositol phos-

Phytase Exhibiting PTPase Activity
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7471
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Received for review February 27, 2008. Revised manuscript received
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