Báo cáo khoa học: Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1 pptx

Site-directed mutagenesis, kinetic and inhibition studies
of aspartate ammonia lyase from Bacillus sp. YM55-1
Vinod Puthan Veetil
1
, Hans Raj
1
, Wim J. Quax
1
, Dick B. Janssen
2
and Gerrit J. Poelarends
1
1 Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands
2 Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands
Aspartate ammonia lyases (also referred to as asparta-
ses) are microbial enzymes that catalyze the reversible
deamination of l-aspartate (1) to yield fumarate (2)
and ammonia (3) (Scheme 1). These enzymes have
been purified and characterized from a number of
Gram-positive and Gram-negative bacteria, including
Escherichia coli, Hafnia alvei, Pseudomonas fluores-
cens, Bacillus subtilis and Bacillus sp. YM55-1 [1–11].
Keywords
aspartase; aspartate ammonia lyase;
Bacillus; deamination; enzyme mechanism
Correspondence
G. J. Poelarends, Department of
Pharmaceutical Biology, Groningen
Research Institute of Pharmacy, University
of Groningen, Antonius Deusinglaan 1, 9713
AV Groningen, The Netherlands

Fax: +31 50 3633000
Tel: +31 50 3633354
E-mail:
(Received 23 January 2009, revised 6 March
2009, accepted 20 March 2009)
doi:10.1111/j.1742-4658.2009.07015.x
Aspartate ammonia lyases (also referred to as aspartases) catalyze the revers-
ible deamination of l-aspartate to yield fumarate and ammonia. In the pro-
posed mechanism for these enzymes, an active site base abstracts a proton
from C3 of l-aspartate to form an enzyme-stabilized enediolate intermediate.
Ketonization of this intermediate eliminates ammonia and yields the prod-
uct, fumarate. Although two crystal structures of aspartases have been deter-
mined, details of the catalytic mechanism have not yet been elucidated. In
the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142,
Thr187, His188, Lys324 and Asn326) were mutated in the structurally char-
acterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a
model of the complex in which l-aspartate was docked manually into the
active site of AspB, the residues responsible for binding the amino group of
l-aspartate were predicted to be Thr101, Asn142 and His188. This postulate
is supported by the mutagenesis studies: mutations at these positions resulted
in mutant enzymes with reduced activity and significant increases in the K
m
for l-aspartate. Studies of the pH dependence of the kinetic parameters of
AspB revealed that a basic group with a pK
a
of approximately 7 and an
acidic group with a pK
a
of approximately 10 are essential for catalysis.
His188 does not play the typical role of active site base or acid because the

H188A mutant retained significant activity and displayed an unchanged pH-
rate profile compared to that of wild-type AspB. Mutation of Ser140 and
Thr141 and kinetic analysis of the mutant enzymes revealed that these resi-
dues are most likely involved in substrate binding and in stabilizing the
enediolate intermediate. Mutagenesis studies corroborate the essential role
of Lys324 because all mutations at this position resulted in mutant enzymes
that were completely inactive. The substrate-binding model and kinetic anal-
ysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in
binding the C1 carboxylate group of the substrate. A catalytic mechanism
for AspB is presented that accounts for the observed properties of the
mutant enzymes. Several features of the mechanism that are also found in
related enzymes are discussed in detail and may help to define a common
substrate binding mode for the lyases in the aspartase ⁄ fumarase superfamily.
Abbreviations
Ap, ampicillin; AspA, aspartase from E. coli; AspB, aspartase from Bacillus sp. YM55-1; FumC, fumarase C from E. coli; RCSB, Research
Collaboratory for Structural Bioinformatics; PDB, Protein Data Bank.
2994 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
The best studied example is the aspartase (AspA) from
E. coli, for which the crystal structure has been eluci-
dated [12]. AspA functions as a homotetramer, where
each monomer consists of 478 amino acid residues,
and is allosterically activated by its substrate ( 1) and
Mg
2+
ions, which are required for activity at alkaline
pH [3,13]. The enzyme has a rather narrow substrate
specificity; it is specific for 1 and 2, and only hydroxyl-
amine can substitute for 3 as a substrate [14–16].
Stereochemical, kinetic isotope and pH-rate studies
indicate that AspA catalyzes an anti-elimination reac-

tion, where an active site base (with a pK
a
of approxi-
mately 6.5) abstracts the proton from C3 of 1 to form
an enzyme-stabilized enediolate intermediate (4;
Scheme 2) [5,6,16–18]. The proposed enediolate inter-
mediate (i.e. aci-carboxylate) can rearrange to elimi-
nate ammonia (3) and form the product, fumarate (2).
The rate determining step is the cleavage of the
carbon-nitrogen bond, which may be facilitated by a
general acid that protonates the leaving ammonia
group. Additional support for the formation of an
enzyme-stabilized enediolate as an intermediate during
the deamination reaction is provided by inhibition
studies demonstrating that 3-nitro-2-aminopropionate,
when present in its resonance-stabilized nitronate
(aci-form) state, binds very tightly to AspA as a
transition state analog [19]. On the basis of the crystal
structure of AspA and site-directed mutagenesis
studies, two positively charged residues (Arg29 and
Lys327) were proposed to bind the two carboxylate
groups of 1 (C4 and C1, respectively) [12,20,21]. It was
further postulated that Ser143 functions as the general
acid catalyst [21].
On the basis of these studies, a picture of the cata-
lytic mechanism of AspA has emerged [16]. However,
this is far from complete and major issues remain
unresolved. One issue concerns the identity of the gen-
eral base catalyst that abstracts the C3 proton, and
that of other essential catalytic and substrate-binding

residues. Another issue concerns whether substrate
binding induces a conformational change that moves
other residues into the active site, as might be expected
for an enzyme that is allosterically activated by its sub-
strate. The crystal structure of AspA does not address
these questions because it was solved in the absence of
a bound ligand [12]. Attempts to obtain a crystal struc-
ture of AspA (or any other aspartase) complexed with
substrate, product or a competitive inhibitor have thus
far proved unsuccessful.
In our studies, we focus on aspartase (AspB) from
the thermophilic bacterium Bacillus sp. YM55-1
[10,11]. This aspartase is of considerable biocatalytic
interest because of its high activity and enantioselec-
tivity, relative thermostability and lack of allosteric
regulation by substrate or metal ions [10,11]. It also
efficiently catalyzes the reverse reaction (i.e. the
addition of ammonia to fumaric acid). Moreover, the
broad nucleophile specificity of AspB enables the use
of a range of alternative nucleophiles, such as methyl-
amine, hydroxylamine, hydrazine and methoxylamine,
in the conjugate addition reaction [22]. These proper-
ties indicate that AspB is a promising biocatalyst for
the synthesis of enantiopure N-substituted aspartic
acids.
The crystal structure of AspB (also in the absence of
a bound ligand) was recently solved (Fig. 1) and shows
that the overall topology and active site architecture of
AspB are similar to those observed in AspA and fuma-
rase C (FumC) from E. coli, confirming its member-

ship in the aspartase ⁄ fumarase superfamily of enzymes
[23]. Similar to AspA, AspB functions as a homotetr-
amer, but now each subunit is composed of 468 amino
acid residues [11,23]. The functional tetramer contains
four active sites, each harboring residues from three
different subunits [23]. A structural model of the com-
plex in which l-aspartate was docked manually into
the active site of AspB suggested interactions that
might be responsible for the binding and activation
–
O
2
C
CO
2
–
–
O
2
C
CO
2
–
NH
3
NH
3
+
+
123

Scheme 1. Reversible deamination of L-aspartate catalyzed by
aspartase.
O
–
O
–
O
2
C
NH
3
H H
B:
O
–
O
–
–
O
2
C
NH
3
O
–
O
–
O
2
C

NH
3
+
Arg-29
Lys-327
Ser-143
+
+
1423
Scheme 2. A schematic representation of the catalytic mechanism of AspA
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 2995
(i.e. polarization of the C4 carboxylate group) of the
substrate [23]. It is noteworthy that this approach of
manual docking of the substrate in the active site,
which may provide valuable insight into how residues
at the active site interact with the substrate, is compli-
cated for AspA because this enzyme is allosterically
activated by its substrate [13]. Hence, it is reasonable
to expect some significant differences between the
active site structure of the apoenzyme and that of
the enzyme–substrate complex. In the docking model,
the C1 carboxylate group of l-aspartate forms hydro-
gen-bonding interactions with the hydroxyl group of
Thr187, the amino group of Lys324 and the amide
group of Asn326, whereas the C4 carboxylate group of
the substrate forms hydrogen bonds with the hydroxyl
groups of Ser140 and Thr141 [23]. According to the
model, the amino group of l-aspartate forms hydro-
gen-bonding interactions with the side chains of

Thr101, Asn142 and His188 [23]. In the present study,
we performed site-directed mutagenesis experiments
on all of these residues to provide further insight into
the mechanism for the AspB-catalyzed deamination
reaction. The results obtained also have implications
for our understanding of the catalytic mechanism of
AspA.
Results
pH dependence of the kinetic parameters of AspB
The pH dependences of k
cat
and k
cat
⁄ K
m
for the AspB-
catalyzed deamination of l-aspartate (1; Scheme 1)
were determined in 100 mm sodium phosphate buffer
over the pH range 6.0–10.0 at 37 °C. Both parameters
show a bell-shaped dependence on pH, with limiting
slopes of unity on either side of the pH maximum,
indicating that both a basic group (ascending limb)
and acidic group (descending limb) are important for
catalysis. The pH dependences of k
cat
and k
cat
⁄ K
m
are

given by Eqns (1,2):
k
cat ðpHÞ
¼ðk
cat
Þ
max
=ð1 þ½H
þ
=K
1
þ K
2
=½H
þ
Þ ð1Þ
k
cat
=K
mðpHÞ
¼ðk
cat
=K
m
Þ
max
=ð1 þ½H
þ
=K
1

þK
2
=½H
þ
Þ ð2Þ
where K
1
is the ionization constant of the basic group
and K
2
is the ionization constant of the acidic group,
with both being important for catalysis [24].
A nonlinear least-squares fit of the pH-dependence
of log (k
cat
⁄ K
m
), which follows the ionizations in the
free enzyme and the free substrate, to the logarithmic
form of Eqn (2) gives pK
a
values of pK
1
= 7.1 ± 0.1
and pK
2
= 9.8 ± 0.2 (Fig. 2). A nonlinear least-
squares fit of the pH-dependence of log (k
cat
), which

follows the ionizations in the enzyme–substrate com-
plex, to the logarithmic form of Eqn (1) yields pK
a
values of pK
1
= 6.2 ± 0.4 and pK
2
= 11.3 ± 2 (data
not shown). Because data could not be collected at
high enough pH values to clearly define the descending
limb (i.e. the extension of these studies beyond pH 10
is precluded by enzyme denaturation), the pK
a
value
Fig. 1. A close-up of the active site of AspB [23]. The roles of the
key active site residues (Thr101, Ser140, Thr141, Asn142, Thr187,
His188, Lys324 and Asn326) and their interactions are discussed in
the text. Suffixes A, B and C indicate that the residues originate
from three different subunits. Prepared using
PYMOL [39].
Fig. 2. pH-dependence of log k
cat
⁄ K
m
for the deamination of
L-aspartate (1) by wild-type AspB ( ) and the T141A (d) and H188A
(
) mutants. The curves were generated by a nonlinear least-
squares fit of the data to the logarithmic form of Eqn (2). Errors
given in the text are standard deviations.

Mechanism of aspartase from Bacillus sp. YM55-1 V. Puthan Veetil et al.
2996 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
for the descending limb may be somewhat less than
the calculated value.
Construction and purification of the AspB
mutants
The residues selected for mutagenesis in the present
study were Thr101, Ser140, Thr141, Asn142, Thr187,
His188, Lys324 and Asn326 (Fig. 1). Similar to wild-
type AspB, all mutant proteins were constructed as
His6-tagged fusion proteins, produced in E. coli
TOP10, and purified to > 95% homogeneity (as
assessed by SDS ⁄ PAGE) using a Ni-based immobilized
metal affinity chromatography procedure [22]. This
purification procedure is highly specific for His6-tagged
proteins, eliminating the possibility that wild-type and
mutant AspB enzymes isolated from E. coli TOP10 are
contaminated with native aspartase from this host. A
previous study has shown that the recombinant His6-
tagged wild-type AspB has only slightly reduced activ-
ity compared to the native protein (without fusion tag)
[22]. The yield of mutant protein from cell culture
varied in the range 8–20 mgÆL
)1
.
Each AspB mutant was analyzed by nondenaturing
PAGE (data not shown). The mutant enzymes were
found to migrate comparably with the wild-type
enzyme, which suggests that the oligomeric association
of the mutants was still intact and that gross confor-

mational changes are unlikely to be present. The struc-
tural integrity of some mutants was also assessed by
circular dichroism (CD) spectroscopy (see below).
Mutagenesis of Thr101, Asn142 and His188
On the basis of a previously reported structural model
of the complex in which l-aspartate (1) was docked
manually into the active site of AspB, the residues
responsible for binding the amino group of 1 are
predicted to be Thr101, Asn142 and His188 [23]. To
investigate the importance of these residues to the
mechanism of AspB, eight single site-directed mutants
were constructed in which Thr101 was replaced with an
alanine or serine (T101A and T101S), Asn142 with an
alanine or glutamine (N142A and N142Q) and His188
with an alanine, glutamine, lysine or arginine (H188A,
H188Q, H188K and H188R). These mutations are
expected to completely remove the functional side chain
(e.g. T101A) or to replace the side chain with another
one that has a similar functional group (e.g. T101S).
The activities of the mutants were assayed using 1 as
the substrate. It was found that replacement of His188
with a glutamine, lysine or arginine essentially abol-
ished enzymatic activity (Table 1). Under the condi-
tions of the kinetic assays, no activity could be
detected for these mutants. A conservative estimate of
the sensitivity of the assay indicates an at least 10
6
-fold
decrease in k
cat

⁄ K
m
compared to that of wild-type
AspB. Substitution of His188 with an alanine, how-
ever, resulted in an active enzyme with an approxi-
mately 57-fold reduction in k
cat
and a 1.8-fold increase
in K
m
, which results in a approximately 100-fold
decrease in k
cat
⁄ K
m
. Hence, the major effect of this
mutation is on the value of k
cat
.
The mutation of Thr101 to an alanine has a large
effect on the catalytic efficiency of AspB. For the
T101A mutant, a plot of various concentrations of 1
versus the initial rates measured at each concentration
remained linear up to 1 m. Hence, the T101A mutant
could not be saturated. Accordingly, only the k
cat
⁄ K
m
was determined, and this parameter is reduced approx-
imately 7100-fold compared to that of wild-type AspB

(Table 1). The mutation of Thr101 to another residue
with an aliphatic hydroxyl group (serine), however,
has a less drastic effect on k
cat
⁄ K
m
. For the T101S
mutant, which could also not be saturated with 1, the
k
cat
⁄ K
m
is reduced only approximately 80-fold.
Replacement of Asn142 with an alanine resulted in a
mutant enzyme without detectable activity (Table 1),
emphasizing the importance of this residue to the
mechanism of AspB. However, substitution of Asn142
with a glutamine, which also contains a terminal amide
group, resulted in an active enzyme with an approxi-
mately 3000-fold reduction in k
cat
⁄ K
m
.
The T101A, N142A and H188A mutants were ana-
lyzed by CD, and the spectra of these mutants were
comparable to that of wild-type AspB, indicating that
Table 1. Kinetic parameters for the deamination of L-aspartate by
wild-type AspB and the Thr101, Asn142 and His188 mutants.
Steady-state kinetic parameters were determined in 50 m

M sodium
phosphate buffer (pH 8.5) at 25 °C. Errors are standard deviations.
ND, not determined (a conservative estimate of the sensitivity of
the assay indicates an at least 10
6
-fold decrease in k
cat
⁄ K
m
com-
pared to that of wild-type AspB). In those cases where no activity
was detected upon prolonged incubation with substrate, k
cat
values
are given as < 0.001.
Enzyme k
cat
(s
)1
) K
m
(mM) k
cat
⁄ K
m
(M
)1
Æs
)1
)

Wild-type 40 ± 7 15 ± 2 2.7 · 10
3
T101A > 0.38 > 1000 3.8 · 10
)1
T101S > 34 > 1000 3.4 · 10
1
N142A < 0.001 ND ND
N142Q > 0.88 > 1000 8.8 · 10
)1
H188A 0.7 ± 0.1 27 ± 2 2.6 · 10
1
H188Q < 0.001 ND ND
H188R < 0.001 ND ND
H188K < 0.001 ND ND
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 2997
the mutations did not result in any major conforma-
tional changes (Fig. 3A).
Mutagenesis of Ser140 and Thr141
To investigate the importance of Ser140 and Thr141,
which are the two residues implicated in binding the
C4 carboxylate group of 1 [23], to the mechanism of
AspB, seven single site-directed mutants were con-
structed (S140A, S140R, S140K, T141A, T141V,
T141R and T141K), as well as two double mutants
(S140G ⁄ T141G and S140K ⁄ T141K). Mutation to Gly,
Ala and Val is expected to remove the functional
group, whereas mutation to Arg or Lys may introduce
a new functional group. It was found that replacement
of Ser140 and Thr141 with an arginine or lysine essen-

tially abolished enzymatic activity (Table 2). The
T141K mutant had a low amount of activity (approxi-
mately 40 000-fold reduction in k
cat
⁄ K
m
), whereas the
S140R, S140K and T141R mutants had no detectable
activity. The T141V mutant and the two double
mutants (S140G ⁄ T141G and S140K ⁄ T141K) also were
completely inactive. Substitution of Ser140 and Thr141
by an alanine, however, resulted in active enzymes.
For the T141A mutant, there is a 133-fold decrease in
k
cat
and an approximately nine-fold decrease in K
m
A
B
C
Fig. 3. Far-UV CD spectra of wild-type (Wt) and mutant enzymes.
(A) Superimposed spectra of wild-type AspB and the T101A,
N142A and H188A mutants. (B) Superimposed spectra of wild-type
AspB and the S140A and T141A mutants. (C) Superimposed spec-
tra of wild-type AspB and the T187A, K324A and N326A mutants.
Spectra were measured in 10 m
M NaH
2
PO
4

buffer (pH 8.5) at a
protein concentration of approximately 5 l
M.
Table 2. Kinetic parameters for the deamination of L-aspartate by
wild-type AspB and the Ser140 and Thr141 mutants. Steady-state
kinetic parameters were determined in 50 m
M sodium phosphate
buffer (pH 8.5) at 25 °C. Errors are standard deviations. ND, not
determined.
Enzyme k
cat
(s
)1
) K
m
(mM) k
cat
⁄ K
m
(M
)1
Æs
)1
)
Wild-type 40 ± 7 15 ± 2 2.7 · 10
3
S140A > 40 > 400 1.0 · 10
2
S140R < 0.001 ND ND
S140K < 0.001 ND ND

T141A 0.3 ± 0.01 1.7 ± 0.1 1.8 · 10
2
T141V < 0.001 ND ND
T141R < 0.001 ND ND
T141K > 0.027 > 400 6.8 · 10
)2
S140G ⁄ T141G < 0.001 ND ND
S140K ⁄ T141K < 0.001 ND ND
Mechanism of aspartase from Bacillus sp. YM55-1 V. Puthan Veetil et al.
2998 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
using 1. As a result, the k
cat
⁄ K
m
is reduced 15-fold.
The major effect of this mutation is observed in k
cat
.
For the S140A mutant, only the k
cat
⁄ K
m
could be
determined, and this parameter is reduced 27-fold
compared to that of wild-type AspB. Because the
increase in K
m
for the S140A mutant is > 27-fold, the
k
cat

must be > 40 s
)1
. Hence, the major effect of this
mutation is likely observed in K
m
. The CD spectra of
the S140A and T141A mutants revealed no significant
differences compared to that of the wild-type protein
(Fig. 3B).
Mutagenesis of Thr187, Lys324 and Asn326
The substrate-binding model suggests that residues
Thr187, Lys324 and Asn326 are responsible for bind-
ing the C1 carboxylate group of 1 [23]. To investigate
the importance of these residues for AspB activity, 10
single site-directed mutants were constructed in which
Thr187 was replaced by an alanine or serine (T187A
and T187S), Asn326 with an alanine or glutamine
(N326A and N326Q) and Lys324 with an alanine,
serine, valine, histidine, arginine or aspartic acid
(K324A, K324S, K324V, K324H, K324R and
K324D). It was found that replacement of Lys324
with a small (alanine), polar (serine), charged (histi-
dine, arginine and aspartate) or hydrophobic (valine)
residue resulted in mutant enzymes with no detectable
activity (Table 3), emphasizing the essential role of
Lys324 in the mechanism of AspB. Prolonged incuba-
tion with 1 revealed a small amount of activity for
the K324R mutant, whereas all other mutants had no
detectable activity. The activity of the K324R mutant
is too low to measure kinetic parameters. Mutations

at positions Thr187 and Asn326 resulted in active
enzymes. Substitution of Thr187 with a serine resulted
in a mutant (T187S) with a surprisingly improved k
cat
(approximately five-fold) compared to that of wild-
type AspB. The K
m
value, however, increased signifi-
cantly (approximately 11-fold). As a result, the
k
cat
⁄ K
m
is reduced approximately 2.3-fold. For the
T187A, N326A and N326Q mutants, only the k
cat
⁄ K
m
values could be determined, and these values are
reduced 6280-fold, 22 500-fold and 168 750-fold,
respectively, compared to the k
cat
⁄ K
m
of wild-type
AspB.
The T187A, K324A, and N326A mutants were also
analyzed by CD and showed no detectable differences
from the wild-type protein (Fig. 3C).
pH dependence of the kinetic parameters of the

T141A and H188A mutants
To determine whether Thr141 or His188 was responsi-
ble for the ascending limb (i.e. the basic group on the
enzyme important for catalysis) or descending limb
(i.e. the acidic group on the enzyme important for
catalysis) of the pH-rate profile of wild-type AspB, the
pH dependence of k
cat
⁄ K
m
for the T141A- and
H188A-catalyzed deamination of 1 was determined
over the pH range 6.0–10. Similar to wild-type AspB,
the pH-rate profiles of the T141A and H188A mutants
(comprising the only mutations at these positions that
resulted in an active enzyme) are bell-shaped with
slopes of unity (Fig. 2). For the T141A mutant, pK
a
values of pK
1
= 6.8 ± 0.1 and pK
2
= 11.1 ± 0.5
were found. For the H188A mutant, pK
a
values of
pK
1
= 7.4 ± 0.3 and pK
2

= 9.4 ± 0.3 were found.
Competitive inhibition of AspB and the T141A
and H188A mutants
It has previously been reported that 3-nitropropionate
and d-malate are competitive inhibitors of the deami-
nation activity of AspA with K
i
values of 0.83 and
0.66 mm, respectively [14]. These observations
prompted us to examine whether these compounds are
also competitive inhibitors of AspB. Lineweaver–Burk
reciprocal plots using three or four different inhibitor
concentrations demonstrate that 3-nitropropionate and
d-malate are competitive inhibitors of the aspartase
activity of AspB with K
i
values of 2.0 ± 0.5 mm and
68±12mm, respectively. These compounds are also
competitive inhibitors of the aspartase activity of the
T141A and H188A mutants. For the T141A mutant,
K
i
values of 0.5 ± 0.1 mm and 48 ± 12 mm were
found for 3-nitropropionate and d-malate, respectively.
Table 3. Kinetic parameters for the deamination of L-aspartate by
wild-type AspB and the Thr187, Lys324 and Asn326 mutants.
Steady-state kinetic parameters were determined in 50 m
M sodium
phosphate buffer (pH 8.5) at 25 °C. Errors are standard deviations.
ND, not determined.

Enzyme k
cat
(s
)1
) K
m
(mM) k
cat
⁄ K
m
(M
)1
Æs
)1
)
Wild-type 40 ± 7 15 ± 2 2.7 · 10
3
T187A > 0.43 > 1000 4.3 · 10
)1
T187S 190 ± 7 163 ± 15 1.2 · 10
3
K324A < 0.001 ND ND
K324S < 0.001 ND ND
K324V < 0.001 ND ND
K324H < 0.001 ND ND
K324D < 0.001 ND ND
K324R < 0.001 ND ND
N326A > 0.12 > 1000 1.2 · 10
)1
N326Q > 0.016 > 1000 1.6 · 10

)2
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 2999
For the H188A mutant, K
i
values of 0.01 ± 0.001 mm
and 0.4 ± 0.02 mm were found for 3-nitropropionate
and d-malate, respectively (Fig. 4). The results show
that replacing the histidine at position 188 with alanine
results in a mutant with a surprisingly improved affin-
ity for 3-nitropropionate (approximately 200-fold) and
d-malate (approximately 170-fold).
Discussion
On the basis of sequence analysis and crystallographic
observations with AspA [Research Collaboratory for
Structural Bioinformatics (RCSB) Protein Data Bank
(PDB) entry 1JSW], AspB (RCSB PDB entry 1J3U)
and FumC (in complex with a substrate analog, cit-
rate; RCSB PDB entry 1FUO) [12,25], Fujii et al. [23]
have made a structural model of the complex in which
l-aspartate (1; Scheme 3) was docked manually into
the active site of AspB. Guided by this substrate-bind-
ing model, we have selected eight active site residues
for mutagenesis to provide further insight into the
mechanism of AspB. In addition, we have performed
pH-rate and inhibition studies to further illuminate the
mechanistic role of certain residues. The results
obtained in these studies are interpreted and related to
the mechanisms of AspA and other superfamily
enzymes.

We first determined the probable chemical mecha-
nism of the AspB-catalyzed reaction and pK
a
values of
potential acid and base catalysts by pH-rate studies.
From the ascending limb of the pH-rate profile of
AspB, a residue with a pK
a
of 7.1 ± 0.1, which must
be deprotonated for optimal activity, may be the gen-
eral base catalyst involved in abstraction of the C3
proton of 1. From the descending limb, a residue with
apK
a
of 9.8 ± 0.2, which must be protonated for
optimal activity, may be the general acid catalyst
A
B
Fig. 4. Lineweaver–Burk reciprocal plots showing the competitive
mode of inhibition of the H188A mutant by 3-nitropropionate (A)
and
D-malate (B). The curves were generated by fitting the data by
nonlinear regression analysis using the equation for competitive
inhibition, yielding the K
i
values given in the text.
O
-
ONH
3

H H
B:
O
–
O
–
–
O
2
C
NH
3
+
1
4
Thr-187
Lys- 324
Asn -3 26
Thr-141
T
h
r
-
1
0
1
A
s
n
41

-
2
H
i
s
-
1
8
8
Ser-140
B:H
Thr-141
O
H
Ser-14 0
O
H
HN
N
His-188
Thr-101
OH
+
Asn-142
H
2
N O
Thr-187
Lys -324
Asn-326

O
O
O
H
NH
3
+
NH
2
O
2 + 3
Scheme 3. A schematic representation of the proposed catalytic mechanism of AspB.
Mechanism of aspartase from Bacillus sp. YM55-1 V. Puthan Veetil et al.
3000 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
involved in protonation of the leaving amino group of
1. However, it is important to emphasize that it is
presently unknown whether the protonation state of
the leaving group in the AspB-catalyzed reaction is
that of ammonia or ammonium ion [6,23]. If the
amino group is released as ammonia, a general acid
catalyst may not be required for the reaction. The
descending limbs of the k
cat
⁄ K
m
versus pH and k
cat
versus pH profiles would then reflect the deprotonation
of the amino group of the substrate (the pK
a

of the
amino group of 1 = 9.8) and active-site bound inter-
mediate (i.e. the putative enediolate), respectively.
Hence, the observed pH dependence of the kinetic
parameters for the AspB-catalyzed deamination of 1 is
most simply explained in terms of the ionization of a
single basic group at the active site and the ionization
of the amino group of the substrate (1) or enzyme-
bound intermediate (4) (Scheme 3).
The substrate binding model of AspB suggests that
His188 is one of the residues that interacts, via hydro-
gen-bonding, with the amino group of 1 [23]. Intrigu-
ingly, this histidine residue is conserved in FumC and
other fumarase ⁄ aspartase superfamily members (e.g.
argininosuccinate lyase, d-crystallin and adenylosucci-
nate lyase) but is replaced by Gln191 in AspA [23].
Mutations of the conserved histidine residue severely
impair catalysis in the former enzymes [26–28]. These
results, taken together with crystallographic observa-
tions, have led to several proposals for the catalytic
role of the histidine [29–31]. These include roles for the
histidine as the general base catalyst that abstracts the
proton from C3 of the substrate, the general acid cata-
lyst that protonates the leaving C2 group, or the criti-
cal residue that activates an active site water molecule,
which then functions as the C3 proton abstracting base
[29–31].
To assess its role in catalysis, we have mutated
His188 in AspB. Examination of the kinetic properties
of the H188A mutant, which is the only mutation at

this position that resulted in an active enzyme, shows
that the major effect of this mutation is on the value
of k
cat
. No significant change was observed in the CD
spectrum of this mutant, demonstrating that the loss
of activity resulting from replacement of His188 by
alanine is not a consequence of the loss of structural
integrity of the enzyme. Taken together, these observa-
tions suggest an important role for His188 in catalysis.
However, the pH-rate profile of the H188A mutant
has the same overall shape as that of wild-type AspB,
with pK
a
values of approximately 7.4 and 9.4, suggest-
ing that the protonation or deprotonation of His188 is
not responsible for the observed loss of activity on
either side of the pH optimum. Therefore, we conclude
that His188 does not function as the typical general
acid or general base catalyst in AspB, which is consis-
tent with the absence of this residue in AspA.
One potential explanation for the loss in activity of
the H188A mutant is that His188 could position and
‘lock’ the amino group of 1 in a favorable orientation
for deamination. In this scenario, loss in activity of the
H188A mutant is a result of the removal of optimal
hydrogen-bonding interactions with the amino func-
tionality, locating this group in an unfavorable posi-
tion for the elimination reaction. Support for this view
is provided by an analysis of the substrate specificity

of the H188A mutant in the reverse amine addition
reaction. In comparison to the wild-type AspB-cata-
lyzed addition of methylamine to fumarate, the H188A
mutant displays a two-fold increase in k
cat
and a
higher K
m
for methylamine, suggesting that His188 is
one of the residues that influences the nucleophile (i.e.
amine) specificity of AspB (V. Puthan Veetil &
G. J. Poelarends, unpublished results). Extrapolation
of this observation to substrate binding supports the
proposed role for His188 in binding the amino group
of 1 (Scheme 3). The corresponding glutamine residue
in AspA (Gln191) may participate in a similar hydro-
gen-bonding interaction with the substrate. Another
notable effect of the H188A mutant is the 170–200-
fold increase in the binding affinity for the competitive
inhibitors 3-nitropropionate and d-malate. This sug-
gests that the decrease in catalysis for this mutant may
be the result of a combination of effects, including the
suboptimal positioning of the amino group and the
slower release of product.
In the substrate binding model of AspB, two other
residues (Thr101 and Asn142) are implicated in bind-
ing the amino group of 1 [23]. The corresponding res-
idues (Thr100 and Asn141) in the FumC–citrate
complex were found to assist His188 (corresponding
to His188 in AspB) in binding an active site water

molecule [23,25]. This geometry suggests that these
three residues may be responsible for the positioning
and activation of the nucleophilic water molecule in
the FumC-catalyzed hydration reaction, as well as the
positioning and protonation of the leaving hydroxyl
group in the reverse dehydration reaction. To exam-
ine the role of Thr101 and Asn142 in AspB, we have
mutated these two residues. Complete removal of the
functionality at position Thr101 by replacement with
an alanine leads to a significant increase in K
m
(> 66-fold) and a large decrease in catalytic efficiency
(> 7000-fold). The same substitution at position
Asn142 even results in a complete loss of activity.
Both these alanine mutants retain their overall struc-
tural integrity, as assessed by CD spectroscopy. These
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 3001
observations, coupled with the enhancement in activ-
ity observed with a serine (position Thr101) or gluta-
mine (position Asn142) mutation at these positions,
suggest that the most likely role for Thr101 and
Asn142 is to participate in a hydrogen-bonding inter-
action with the amino group of 1 (Scheme 3). Simi-
larly, the corresponding residues in AspA (Thr104
and Asn145), which are positionally conserved but
have slighty different side chain orientations [12,23],
may assist Gln191 in binding the amino group of the
substrate.
In the substrate binding model of AspB, the hydro-

xyl groups of Ser140 and Thr141 are hydrogen bonded
to the C4 carboxylate group of 1 [23]. This suggests
roles for the hydroxyl functional groups of Ser140 and
Thr141 in binding the substrate, and possible interme-
diates, in the reaction mechanism of AspB. Crystallo-
graphic studies on FumC support this view [25]. In the
structure of the FumC–citrate complex, Ser139 and
Ser140 (corresponding to Ser140 and Thr141 in AspB)
are hydrogen bonded to one of the carboxylate groups
of citrate [23,25]. This geometry suggests the impor-
tance of these residues in binding one of the two
carboxylate groups of the substrate. In AspB, Ser140
and Thr141 are located at the N-terminal end of
a-helix 6 in the positively charged environment created
by a dipole moment of this helix [23].
In the present study, Ser140 and Thr141 in AspB
were mutated to assess their role in catalysis. The best
characterized mutants are the S140A and T141A
mutants, which have measurable activity and show no
significant differences in their CD spectra compared to
that of the wild-type AspB. Examination of the kinetic
properties for the S140A mutant shows that there is a
small effect on k
cat
⁄ K
m
and a larger increase in K
m
,
suggesting that Ser140 plays a major role in binding

the C4 carboxylate group of 1 (Scheme 3). By contrast
to these observations, examination of the kinetic prop-
erties for the T141A mutant shows that the major
effect of replacing Thr141 by alanine is on the value of
k
cat
. This suggests an important role for Thr141 in
catalysis. Another notable effect of the Thr141 muta-
tion is the increase in the binding affinity for the sub-
strate (assuming that the K
m
reflects substrate binding)
and for the competitive inhibitors 3-nitropropionate
and d-malate.
A major part of the loss in activity of the T141A
mutant is likely a result of the removal of optimal
hydrogen-bonding interactions with the proposed eno-
late anion intermediate formed at the C4 carbonyl
position of 1, making the abstraction of a proton
from the C3 position less favorable. Whether the
intermediate is an enolate anion (i.e. a resonance-
stabilized carbanion) derived by abstraction of the C3
proton from 1, or an enol that could be obtained by
protonation of an enolate anion, remains unknown.
Presumably, Thr141, together with Ser140, polarizes
the C4 carboxylate group and stabilizes the enediolate
intermediate (4) formed upon abstraction of the C3
proton (Scheme 3). This proposed role for Thr141 in
the mechanism of AspB shares similarity with the one
proposed for Glu317 in mandelate racemase [32]. This

enzyme catalyzes the equilibration of the (R)- and
(S)-enantiomers of mandelate. On the basis of muta-
genesis, crystallographic and kinetic isotope studies,
Mitra et al. [32] proposed that Glu317 in mandelate
racemase functions as a general acid catalyst in the
concerted general acid–general base catalyzed forma-
tion of a stabilized enolic tautomer of mandelic acid
as a reaction intermediate. Glu317 can function as a
general acid catalyst because it is protonated when
substrate binds at the active site and it is properly
positioned for partial proton transfer to the carboxyl-
ate group of mandelic acid to form a strong hydro-
gen-bonded enolic intermediate. However, it is clear
from the pH-rate profile of the T141A mutant of
AspB that Thr141 is not the acidic group responsible
for the descending limb of the pH-rate profile of
wild-type AspB. Therefore, we conclude that the
protonation state of Thr141 does not show up in
the pH versus k
cat
⁄ K
m
profile of AspB and that the
descending limb of this profile is caused by deproto-
nation of either the substrate or an unknown acidic
group on the enzyme important for catalysis. The
observation that electrophilic catalysis by Thr141 in
AspB (i.e. there is a 133-fold drop in the value of k
cat
for the T141A mutant) is not as important as that by

Glu317 in mandelate racemase (i.e. there is a 4500-
fold drop in the value of k
cat
for the E317Q mutant)
[32] could be explained, at least in part, by the pres-
ence of the dipole moment of a-helix 6, which likely
assists Thr141 in stabilizing the negative charge that
develops on one of the C4 carboxylate oxygens upon
proton abstraction [23].
A comparison of the crystal structures of AspA and
AspB shows that Ser140 and Thr141 of AspB are posi-
tionally conserved as Ser143 and Thr144 in AspA
(although the side-chain orientations are slightly
different) [23]. Replacement of Ser143 with glycine or
threonine caused a significant decrease in k
cat
(10- and
100-fold, respectively) and a three- to four-fold
increase in K
m
using 1 [21]. On the basis of these
observations, Ser143 in AspA was proposed to func-
tion as the general acid catalyst that protonates the
leaving C2 amino group (Scheme 2) [21]. In view of
the crystallographic observations with FumC [25] and
Mechanism of aspartase from Bacillus sp. YM55-1 V. Puthan Veetil et al.
3002 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
the substrate-binding model of AspB [23], and taken
together with the mutagenesis results of the present
study, it is doubtful that Ser143 functions as the

general acid catalyst in AspA. However, in the absence
of a crystal structure of AspA in complex with
substrate (or a substrate analog), we can only speculate
that the mechanistic roles of Ser143 and Thr144 in
AspA share similarity with those proposed in the pres-
ent study for Ser140 and Thr141 in AspB (Scheme 3).
In the structure of the FumC–citrate complex, one
of the other carboxylate groups of citrate interacts
with the side chain of Lys324 (corresponding to
Lys327 in AspA and Lys324 in AspB) [23,25]. Previ-
ous studies have shown that mutation of Lys327 in
AspA results in a six-fold increase in K
m
and a large
(> 300-fold) decrease in k
cat
[20]. These observations
suggest a role for the lysine residue in binding one of
the two carboxylate groups of the substrate. Accord-
ing to the substrate-binding model of AspB, Lys324
binds the C1 carboxylate group of 1 [23]. The stron-
gest support for this orientation of substrate in the
active site comes from the observation that substitu-
tion of Lys327 in AspA with an asparagine changes
the substrate specificity of the enzyme and allows it to
process l-aspartate-a-amide [33]. Our experiments
clearly show that Lys324 is an essential residue in the
AspB-catalyzed reaction because mutations at this
position result in a complete loss of activity. More-
over, the mutant enzymes appear to retain an intact

overall structural integrity (as shown for the K324A
mutant by CD spectroscopy). It is therefore reason-
able to conclude that Lys324 is crucial for substrate
binding through an interaction with the C1 carboxyl-
ate group (Scheme 3).
In the region surrounding Lys324 in the structure
of the FumC–citrate complex, there are two residues
(Thr187 and Asn326) that are well positioned to assist
Lys324 in binding one of the citrate carboxylate
groups [25]. A comparison of this structure with those
of AspA and AspB shows that these residues are posi-
tionally conserved as Thr190 and Asn329 in AspA
and Thr187 and Asn326 in AspB [23]. The results
obtained in the present study support a role for
Thr187 and Asn326 in assisting Lys324 to bind and
position the substrate (through interactions with the
C1 carboxylate group of 1) (Scheme 3) because
mutants of these two residues display reduced activity
with large effects on the K
m
for 1. Similar mechanistic
roles may be proposed for the corresponding residues
in AspA.
In summary, the mutagenesis and pH-rate studies
performed in the present study support the substrate-
binding model and initial mechanism reported by
Fujii et al. [23] (Scheme 3), although other mecha-
nisms cannot be ruled out. In this mechanism, an
active site base (with a pK
a

of approximately 7.1)
abstracts the proton from C3 of 1 to form an enedio-
late intermediate (4), which is stabilized by both
Ser140 and Thr141. The interaction of two hydroxyl
functional groups with one carboxylate group is con-
sistent with a dianionic aci-carboxylate intermediate.
The identity of the residue with the pK
a
of 7.1 is not
known. (It has been suggested previously [23] that
Ser318 may function as the general base in AspB.
However, this residue is not located in the presumed
active site of the enzyme. To establish whether the
loop containing Ser318 may undergo a large confor-
mational change upon substrate binding, positioning
Ser318 in the vicinity of C3 of the substrate, we have
initiated studies that aim to solve the X-ray structure
of the enzyme–substrate complex.) In the next step,
the enediolate intermediate collapses and eliminates
ammonia (3) to form the fumarate (2) product.
Thr101, Asn142 and His188 could position and ‘lock’
the amino group in a favorable orientation for deami-
nation, whereas Thr187, Lys324 and Asn326 bind the
C1 carboxylate group of the substrate. Strong support
for this arrangement in AspB (Scheme 3) also comes
from the interactions observed in the recently solved
crystal structure of the fumarase ⁄ aspartase superfam-
ily enzyme adenylosuccinate lyase complexed with
adenylosuccinate [31]. In the structure of this com-
plex, Thr122 and Ser123 interact with the d-carboxyl-

ate group (on which the negative charge accumulates
in the aci-carboxylate intermediate) of the succinyl
moiety of adenylosuccinate, whereas Thr170, Lys301
and Asn303 interact with the c-carboxylate group.
The positional conservation of these carboxylate bind-
ing residues (where Ser140, Thr141, Thr187, Lys324
and Asn326 in AspB are replaced by Thr122, Ser123,
Thr170, Lys301 and Asn303 in adenylosuccinate lyase)
suggests a similar mode of substrate binding for these
two superfamily members.
The positional conservation of seven out of eight
active site residues also suggests a mechanism for AspA
that largely parallels that proposed for AspB (Scheme 3)
where the residues in AspB (Thr101, Ser140, Thr141,
Asn142, Thr187, Lys324 and Asn326) are replaced by
the corresponding ones in AspA (Thr104, Ser143,
Thr144, Asn145, Thr190, Lys327 and Asn329). This
proposed mechanism needs to be corroborated by
future crystallographic studies. The utility of 3-nitropro-
prionate as a potent competitive inhibitor and potential
crystallographic ligand for AspB could help to identify
the additional features that are necessary for a fully
active and specific aspartase.
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 3003
Experimental procedures
Materials
l-Aspartic acid, either the free acid or the sodium salt, and
l-arabinose were purchased from Sigma-Aldrich Chemical
Co. (St Louis, MO, USA). Ingredients for buffers and

media were obtained from Duchefa Biochemie (Haarlem,
The Netherlands) or Merck (Darmstadt, Germany). Molec-
ular biology reagents, including restriction enzymes, PCR
reagents, T4 DNA ligase, DNA ladders, agarose and pro-
tein molecular weight standards, were obtained from
F. Hoffman-LaRoche, Ltd. (Basel, Switzerland), Promega
Corp. (Madison, WI, USA), Invitrogen Corp. (Carlsbad,
CA, USA), Finnzymes (Espoo, Finland) or New England
Biolabs (Ipswich, MA, USA). PCR purification, gel extrac-
tion, and Miniprep kits were provided by Macherey-Nagel
(Du
¨
ren, Germany). Pre-packed PD-10 Sephadex G-25 col-
umns were purchased from GE Healthcare Bio-Sciences AB
(Uppsala, Sweden). Oligonucleotides for DNA amplifica-
tion were synthesized by Operon Biotechnologies (Cologne,
Germany).
Bacterial strains, plasmids and growth conditions
Escherichia coli strains DH10B (Invitrogen Corp.) and
XL1-Blue (Stratagene, La Jolla, CA, USA) were used for
cloning and isolation of plasmids. Escherichia coli strain
TOP10 (Invitrogen Corp.) was used in combination with
the pBAD ⁄ Myc-His A vector (Invitrogen Corp.) for recom-
binant protein production. Plasmid pUCBA [11], the DNA
source for the aspB gene, was a kind gift from Y. Kawata
(Department of Biotechnology, Tottori University, Japan).
Escherichia coli cells were grown at 37 °C in LB media.
When required, Difco agar (15 gÆL
)1
), ampicillin (Ap;

100 lgÆmL
)1
) and ⁄ or arabinose (0.04% w ⁄ v) were added to
the medium.
General methods
Techniques for restriction enzyme digestions, ligation, trans-
formation and other standard molecular biology manipula-
tions were based on previously described methods [34] or
carried out in accordance with the manufacturer’s instruc-
tions. The PCR was carried out in a DNA thermal cycler
(model GS-1) obtained from Biolegio (Nijmegen, The Neth-
erlands). DNA sequencing was performed by ServiceXS (Lei-
den, The Netherlands) or Macrogen (Seoul, Korea). Protein
was analyzed by PAGE under either denaturing conditions
using SDS or native conditions on gels containing 7.5–10%
polyacrylamide. The gels were stained with Coomassie bril-
liant blue. Protein concentrations were determined by the
method of Waddell [35]. Kinetic data were obtained on a
V-650 spectrophotometer obtained from Jasco (IJsselstein,
The Netherlands). The cuvettes were mixed using a stirr ⁄ add
cuvette mixer (Bel-Art Products, Pequannock, NJ, USA).
The kinetic data were fitted by nonlinear regression analysis
using grafit (Erithacus, Software Ltd, Horley, UK)
obtained from Sigma-Aldrich Chemical Co. The CD spectra
were recorded on a model 62A-DS spectropolarimeter from
AVIV Biomedical, Inc (Lakewood, NJ, USA).
Construction of AspB mutants
Two standard mutagenesis methods were used to introduce
site-specific mutations into the aspB gene [36,37]. Most
mutants were generated by the overlap extension PCR

method using plasmid pUCBA as the template [36]. The
final PCR products were gel purified, digested with NcoI
and HindIII restriction enzymes, and ligated in frame with
both the initiation ATG start codon and the sequence that
codes for the polyhistidine region of the expression vector
pBAD ⁄ Myc-His A. A few mutants were generated by the
QuikChange mutagenesis method (Stratagene, La Jolla,
CA, USA) using plasmid pBAD (AspB-His), which encodes
His6-tagged wild-type AspB [22], as the template [37]. All
mutant genes were completely sequenced (with overlapping
reads) to verify that only the intended mutation had been
introduced.
Expression and purification of AspB wild-type
and mutants
The AspB enzyme, either wild-type or mutant, was pro-
duced in E. coli TOP10 using the pBAD expression system.
Fresh TOP10 cells containing the appropriate expression
plasmid were collected from a LB ⁄ Ap plate using a sterile
loop and used to inoculate 10 mL of LB ⁄ Ap medium. After
overnight growth at 37 °C, a sufficient quantity of the cul-
ture was used to inoculate 1 L of LB ⁄ Ap medium in a 5 L
Erlenmeyer flask to an initial A
600
of approximately 0.02.
Cultures were grown until A
600
of 0.4–0.6 was reached at
37 °C with vigorous shaking and then induced with arabi-
nose [0.04% (w ⁄ v) final concentration]. Incubation was
continued for 10–12 h at 37 °C. Cells were harvested by

centrifugation (6000 g for 15 min) and stored at )20 °C
until further use.
In a typical purification experiment, cells of a 1 L culture
were thawed and suspended in 10 mL of lysis buffer
(50 mm NaH
2
PO
4
, 300 mm NaCl, 10 mm imidazole,
pH 8.0). Cells were disrupted by sonication for 4 · 1 min
(with 4–6 min of rest in between each cycle) at a 60 W out-
put, after which unbroken cells and debris were removed
by centrifugation (10 000 g for 30 min). The supernatant
was filtered through a 0.45 lm pore diameter filter and
incubated with Ni-nitrilotriacetic acid (1 mL slurry in a
small column at 4 °C for ‡ 18 h), which had previously
been equilibrated with lysis buffer. The nonbound proteins
Mechanism of aspartase from Bacillus sp. YM55-1 V. Puthan Veetil et al.
3004 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
were eluted from the column by gravity flow. The column
was first washed with lysis buffer (10 mL) and then with
buffer A (50 mm NaH
2
PO
4
, 300 mm NaCl, 20 mm imidaz-
ole, pH 8.0; 10 mL). Retained proteins were eluted with
buffer B (50 mm NaH
2
PO

4
, 300 mm NaCl, 250 mm imidaz-
ole, pH 8.0; 3.0 mL). Fractions (approximately 0.5 mL)
were analyzed by SDS ⁄ PAGE on gels containing 10%
acrylamide, and those that contained purified aspartase
were pooled and the buffer was exchanged against 50 mm
NaH
2
PO
4
(pH 8.0) using a pre-packed PD-10 Sephadex
G-25 gelfiltration column. The purified enzyme was stored
at 4 °Cor)80 °C until further use.
CD spectroscopy
Circular dichroism spectra of the wild-type protein and the
purified mutants were measured in 10 mm NaH
2
PO
4
buffer
(pH 8.5) at a concentration of approximately 5 lm in a CD
cell with an optical path length of 1.0 mm.
Enzymatic assay
Kinetic assays were performed at 25 °Cin50mm
NaH
2
PO
4
buffer, pH 8.5, observing the increase in absor-
bance at 240 nm corresponding to the formation of fuma-

rate (2)(e = 2530 m
)1
Æcm
)1
) as described previously [22].
An aliquot of AspB, either wild-type or mutant, was diluted
into buffer (20 mL) and incubated for ‡ 30 min at 25 °C.
Subsequently, a 1 mL portion was transferred to a 10 mm
quartz cuvette and the enzyme activity was assayed by the
addition of a small quantity (1–10 lL) of sodium l-aspar-
tate (1) from a stock solution. The stock solutions were
made up in 50 mm NaH
2
PO
4
buffer (pH 8.5). The concen-
trations of 1 used in the assay varied in the range
5–1000 mm.
pH dependence of the kinetic parameters of
AspB, T141A and H188A
The pH dependence of the steady-state kinetic parameters
was determined in 100 mm sodium phosphate buffers with
pH values in the range 5.5–10.0. The buffers were made up
by combining appropriate quantities of 100 mm NaH
2
PO
4
,
100 mm Na
2

HPO
4
and 100 mm Na
3
PO
4
buffers to maintain
constant ionic strength. For each pH value, a sufficient
quantity of enzyme (from a stock solution in 50 mm
NaH
2
PO
4
, pH 8.0) was equilibrated in buffer (20 mL) for
‡ 30 min at 37 °C. The addition of enzyme did not signifi-
cantly change the pH. Subsequently, aliquots (1 mL) were
removed and assayed for activity using concentrations of
l-aspartic acid (1) in the range 5–100 mm. Stock solutions
of 1 were made in 100 mm NaH
2
PO
4
buffer. The pH of the
stock solutions was adjusted to each desired pH value (5.5–
10.0). The volume of substrate added was 20 lL or less in
all experiments. It was not possible to collect data at pH
values above 10 as a result of enzyme denaturation. Conse-
quently, data could not be collected to high enough pH
values to clearly define the descending limb for wild-type
AspB and the T141A mutant. In these cases, the pK

2
value
reported is an estimate obtained from grafit software.
Kinetics of reversible inhibition of AspB, T141A
and H188A by 3-nitropropionate and
D-malate
The reversible inhibition of AspB and the T141A and
H188A mutants was examined using 3-nitropropionate and
d-malate. For each experiment, a small amount of enzyme
was diluted into 15 mL of assay buffer (50 mm NaH
2
PO
4
buffer, pH 8.5). Subsequently, an aliquot of inhibitor from
a stock solution (in 50 mm NaH
2
PO
4
buffer, pH 8.5) was
added to the diluted enzyme solution to yield the approxi-
mate final inhibitor concentration. After approximately
30 min, aliquots (1 mL) of the resulting solution were
removed and assayed using 10–15 concentrations (range
5–100 mm)of1. The final concentrations of the inhibitors
were in the range 0–10 mm. The mode of inhibition was
determined from Lineweaver–Burk reciprocal plots [38].
The inhibition constants (K
i
values) were obtained by
fitting the data by nonlinear regression analysis using the

equation for competitive inhibition provided in the grafit
software.
Acknowledgements
This research was supported financially by The Neth-
erlands Ministry of Economic Affairs and the B-Basic
partner organizations () through
B-Basic, a public–private NWO-ACTS programme.
G. J. P. was supported by grants (VENI 700.54.401
and VIDI 700.56.421) from the Division of Chemical
Sciences of The Netherlands Organisation of Scien-
tific Research (NWO-CW). We thank Dr Yasushi
Kawata (Department of Biotechnology, Tottori
University, Japan) for the kind gift of plasmid
pUCBA and Dr Yasuo Hata (Institute for Chemical
Research, Kyoto University, Japan) for the kind gift
of the PDB file of the homotetrameric AspB struc-
ture. We gratefully acknowledge Gea K. Schuurman-
Wolters (Department of Biochemistry, University of
Groningen) for her assistance in acquiring the CD
spectra. We are also grateful to Jeroen Bonet, Elisa
Hoekstra and Sam Gijsberts for their assistance in
the construction and purification of several AspB
mutants. Finally, we thank Dr Oliver May, Dr
Stefaan de Wildeman, Dr Friso van Assema and Dr
Bernard Kaptein (DSM Geleen, The Netherlands) for
insightful discussions.
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 3005
References
1 Rudolph FB & Fromm HJ (1971) The purification and

properties of aspartase from Escherichia coli. Arch Bio-
chem Biophys 147, 92–98.
2 Suzuki S, Yamaguchi J & Tokushige M (1973) Studies
on aspartase. I. Purification and molecular properties of
aspartase from Escherichia coli. Biochim Biophys Acta
321, 369–381.
3 Takagi JS, Ida N, Tokushige M, Sakamoto H &
Shimura Y (1985) Cloning and nucleotide sequence of
the aspartase gene of Escherichia coli W. Nucleic Acids
Res 13, 2063–2074.
4 Karsten WE, Hunsley JR & Viola RE (1985) Purifica-
tion of aspartase and aspartokinase-homoserine
dehydrogenase I from Escherichia coli by dye-ligand
chromatography. Anal Biochem 147, 336–341.
5 Nuiry II, Hermes JD, Weiss PM, Chen C-Y & Cook
PF (1984) Kinetic mechanism and location of rate-
determening steps for aspartase from Hafnia alvei.
Biochemistry 23, 5168–5175.
6 Yoon M-Y, Thayer-Cook KA, Berdis AJ, Karsten WE,
Schnackerz KD & Cook PF (1995) Acid-base chemical
mechanism of aspartase from Hafnia alvei. Arch
Biochem Biophys 320, 115–122.
7 Takagi JS, Fukunaga R, Tokushige M & Katsuki H
(1984) Purification, crystallization, and molecular
properties of aspartase from Pseudomonas fluorescens. J
Biochem (Tokyo) 96, 545–552.
8 Takagi JS, Tokushige M & Shimura Y (1986) Clon-
ing and nucleotide sequence of the aspartase gene of
Pseudomonas fluorescens. J Biochem (Tokyo) 100,
697–705.

9 Sun D & Setlow P (1991) Cloning, nucleotide sequence,
and expression of the Bacillus subtilis ans operon, which
codes for L-asparaginase and L-aspartase. J Bacteriol
173, 3831–3845.
10 Kawata Y, Tamura K, Yano S, Mizobata T, Nagai J,
Esaki N, Soda K, Tokushige M & Yumoto N (1999)
Purification and characterization of thermostable aspar-
tase from Bacillus sp. YM55-1. Arch Biochem Biophys
366, 40–46.
11 Kawata Y, Tamura K, Kawamura M, Ikei K, Mizobata
T, Nagai J, Fujita M, Yano S, Tokushige M & Yumoto
N (2000) Cloning and over-expression of thermostable
Bacillus sp. YM55-1 aspartase and site-directed muta-
genesis for probing a catalytic residue. Eur J Biochem
267, 1847–1857.
12 Shi W, Dunbar J, Jayasekera MMK, Viola RE &
Farber GK (1997) The structure of L-aspartate
ammonia-lyase from Escherichia coli. Biochemistry 36,
9136–9144.
13 Karsten WE, Gates RB & Viola RE (1986) Kinetic
studies of L-aspartase from Escherichia coli: substrate
activation. Biochemistry 25
, 1299–1303.
14 Falzone CJ, Karsten WE, Conley JD & Viola RE
(1988) L-Aspartase from Escherichia coli: substrate
specificity and the role of divalent metal ions. Biochem-
istry 27, 9089–9093.
15 Emery TF (1963) Aspartase-catalyzed synthesis of
N-hydroxyaspartic acid. Biochemistry 2, 1041–1045.
16 Viola RE (2000) L-Aspartase: new tricks from an old

enzyme. Adv Enzymol Relat Areas Mol Biol 74, 295–
341.
17 Gawron O & Fondy TP (1959) Stereochemistry of the
fumarase and aspartase catalyzed reactions and of the
Krebs cycle from fumaric acid to d-isocitric acid. JAm
Chem Soc 81, 6333–6334.
18 Hanson KR & Havir EA (1972) The enzymic elimina-
tion of ammonia. In The Enzymes, Vol. 3 (Boyer PD
ed.), pp. 75–166. Academic Press, New York, NY.
19 Porter DJT & Bright HJ (1980) 3-Carbanionic substrate
analogues bind very tightly to fumarase and aspartase.
J Biol Chem 255, 4772–4780.
20 Saribas AS, Schindler JF & Viola RE (1994) Mutagenic
investigation of conserved functional amino acids in
Escherichia coli L-aspartase. J Biol Chem 269, 6313–
6319.
21 Jayasekera MMK, Shi W, Farber GK & Viola RE
(1997) Evaluation of functionally important amino acids
in L-aspartate ammonia-lyase from Escherichia coli.
Biochemistry 36, 9145–9150.
22 Weiner B, Poelarends GJ, Janssen DB & Feringa BL
(2008) Biocatalytic enantioselective synthesis of
N-substituted aspartic acids by aspartate ammonia
lyase. Chem Eur J 14, 10094–10100.
23 Fujii T, Sakai H, Kawata Y & Hata Y (2003) Crystal
structure of thermostable aspartase from Bacillus sp.
YM55-1: structure-based exploration of functional
sites in the aspartase family. J Mol Biol 328, 635–
654.
24 Stivers JT, Abeygunawardana C, Mildvan AS,

Hajipour G & Whitman CP (1996) 4-Oxalocrotonate
tautomerase: pH dependences of catalysis and pK
a
values of active site residues. Biochemistry 35, 814–
823.
25 Weaver T & Banaszak L (1996) Crystallographic
studies of the catalytic and a second site in fumarase
C from Escherichia coli. Biochemistry 35, 13955–
13965.
26 Weaver T, Lees M & Banaszak L (1997) Mutations of
fumarase that distinguish between the active site and a
nearby dicarboxylic acid binding site. Protein Sci 6,
834–842.
27 Lee TT, Worby C, Bao Z-Q, Dixon JE & Colman RF
(1999) His
68
and His
141
are critical contributors to the
intersubunit catalytic site of adenylosuccinate lyase of
Bacillus subtilis. Biochemistry 38, 22–32.
28 Patejunas G, Barbosa P, Lacombe M & O¢Brien WE
(1995) Exploring the role of histidines in the catalytic
Mechanism of aspartase from Bacillus sp. YM55-1 V. Puthan Veetil et al.
3006 FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS
activity of duck d-crystallins using site-directed muta-
genesis. Exp Eye Res 61, 151–154.
29 Blanchard JS & Cleland WW (1980) Use of isotope
effects to deduce the chemical mechanism of fumarase.
Biochemistry 19, 4506–4513.

30 Wu C-Y, Lee H-J, Wu S-H, Chen S-T, Chiou S-H &
Chang G-G (1998) Chemical mechanism of the endo-
genous argininosuccinate lyase activity of duck lens
d2-crystallin. Biochem J 333, 327–334.
31 Tsai M, Koo J, Yip P, Colman RF, Segall ML &
Howell PL (2007) Substrate and product complexes of
Escherichia coli adenylosuccinate lyase provide new
insights into the enzymatic mechanism. J Mol Biol 370,
541–554.
32 Mitra B, Kallarakal AT, Kozarich JW, Gerlt JA,
Clifton JG, Petsko GA & Kenyon GL (1995)
Mechanism of the reaction catalyzed by mandelate
racemase: importance of electrophilic catalysis by
glutamic acid 317. Biochemistry 34, 2777–2787.
33 Asano Y, Kira I & Yokozeki K (2005) Alteration of
substrate specificity of aspartase by directed evolution.
Biomol Eng 22, 95–101.
34 Sambrook J, Fritsch EF & Maniatis T (1989)
Molecular Cloning: A Laboratory Manual, 2nd edn.
Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
35 Waddell WJ (1956) A simple ultraviolet spectrophoto-
metric method for the determination of protein. J Lab
Clin Med 48, 311–314.
36 Ho SN, Hunt HD, Horton RM, Pullen JK & Pease
LR (1989) Site-directed mutagenesis by overlap exten-
sion using the polymerase chain reaction. Gene 77,
51–59.
37 Braman J, Papworth C & Greener A (1996) Site-direc-
ted mutagenesis using double-stranded plasmid DNA

templates. Methods Mol Biol 57 , 31–44.
38 Fersht A (1999) Structure and Mechanism in Protein
Science: A Guide to Enzyme Catalysis and Protein
Folding, pp. 112–114. W.H. Freeman and Company,
New York, NY.
39 DeLano WL (2002) The PyMOL Molecular Graphics
System. DeLano Scientific, San Carlos, CA (http://
www.pymol.org).
V. Puthan Veetil et al. Mechanism of aspartase from Bacillus sp. YM55-1
FEBS Journal 276 (2009) 2994–3007 ª 2009 The Authors Journal compilation ª 2009 FEBS 3007