Báo cáo khoa học: Expression, purification and catalytic activity of Lupinus luteus asparagine b-amidohydrolase and its Escherichia coli homolog potx

Expression, purification and catalytic activity of
Lupinus luteus
asparagine b-amidohydrolase and its
Escherichia coli
homolog
Dominika Borek
1,
*, Karolina Michalska
1,
*, Krzysztof Brzezinski
1
, Agnieszka Kisiel
2
, Jan Podkowinski
2
,
David T. Bonthron
3
, Daniel Krowarsch
4
, Jacek Otlewski
4
and Mariusz Jaskolski
1,2
1
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland;
2
Center for Biocrystallographic
Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland;
3
Molecular Medicine Unit, University of

Leeds, UK;
4
Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, Wroclaw University, Poland
We describe the expression, purification, and biochemical
characterization of two homologous enzymes, with amido-
hydrolase activities, of plant (Lupinus luteus potassium-
independent asparaginase, LlA) and bacterial (Escherichia
coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both
enzymes were expressed in E. coli cells, with (LlA) or with-
out (EcAIII) a His-tag sequence. The proteins were purified,
yielding 6 or 30 mgÆL
)1
of cu lture, respectively. The enzymes
are heat- stable up to 60 °C and show both isoaspartyl di-
peptidase and
L
-asparaginase activities. Kinetic parameters
for both enzymatic reactions have been d etermined, showing
that the isoaspartyl peptidase activity is the dominating one.
Despite sequence similarity to aspartylglucosaminidases, no
aspartylglucosaminidase activity could be d etected. Phylo-
genetic analysis demonstrated the relationship of these pro-
teins to other asparaginases and aspartylglucosaminidases
and suggested their classification as N-terminal nucleophile
hydrolases. This is c onsistent with the observed a utocatalytic
breakdown of the immature proteins into two subunits, with
liberation of an N-terminal t hreonine as a potential catalytic
residue.
Keywords: asparaginase; isoaspartyl peptidase; aspartylglu-
cosaminidase; Ntn-hydrolase; glutathione.

L
-Asparaginases (EC 3.5.1.1) are enzymes that catalyze the
hydrolysis of
L
-asparagine to
L
-aspartate and a mmonia.
Using amino acid sequences and biochemical properties as
criteria, e nzymes with asparaginase activit y can be divided
into several families [1]. The two largest and best-charac-
terized f amilies include bacterial- and plant-type asparagin-
ases. The bacterial-type enzymes have been studied for over
30 years [2], mostly because they are important agents in the
therapy o f s ome types of lymphoblastic leukemias [2–6].
Their homologu es a re found in some mammals and in fungi
[7]. The bacterial-type enzymes frequently exhibit other
activities as well, and this family may be significantly larger
than the collection of sequences deposited as asparaginases.
In particular, enzymes such as glutamin-(asparagin)-ases
(EC 3 .5.1.38) [8,9], lysophospholipases (EC 3.1.1.5) [10],
and the a-subunit of Glu-tRNA amidotransferase
(EC 6 .3.5 ) [ 11], can also be considered part of the bacterial
asparaginase family. It has been shown on the basis of
kinetic and structural studies that two conserved amino acid
motifs are responsible for the activity of the above-
mentioned proteins [7,9,12–18].
The plant-type enzymes have been studied less thor-
oughly. In plants,
L
-asparagine is the major nitrogen storage

and transport compound, and it may also accumulate under
stress conditions [19–21]. Asparaginases liberate from
asparagine the ammonia that i s necessary f or protein
synthesis. There are two groups of such proteins, called
potassium-dependent and potassium-independent aspara-
ginases. We have reported previously the i dentification and
sequencing of cDNA (GenBank accession number
GI:4139265) coding for a protein (termed LlA) from yellow
lupin (Lupinus l uteus) that b elongs t o t he potassium-
independent group [22]. Its homologues have been charac-
terized biochemically for some legumes [23–27]. The levels
of expression of these proteins are highest in t he embryo of
developing seeds, when the storage proteins are being
deposited, and start decreasing 45–50 days after anthesis
[19,23]. Also, in plants using ureides for nitroge n transport,
such as soybean during symbiosis with nitrogen fixing
bacteria, the asparginase gene is expressed at low level [23].
Correspondence to M. Jaskolski, Department of Crystallography,
Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6,
60-780 Poznan, Poland. Fax: +48 61 8658008,
Tel.: +48 61 8291274, E-mail:
Abbreviations: EcAIII, Escherichia coli iaaA gene product; GlcNAc-
L
-Asn, N
4
-(b-N-acetylglucosaminyl)-
L
-asparagine; GOT, glutamate-
oxaloacetate transaminase; IPTG, isopropyl thio-b-
D

-galactoside;
LlA, Lupinus luteus asparaginase; MDH, malate dehydrogenase;
ML, maximum likelihood; N-J, neighbor-joining; Ntn, N-terminal
nucleophile; PEG8K, polyethylene glycol 8000.
Enzymes: N
4
-(b-N-acetylglucosaminyl)-
L
-asparaginase (EC 3.5.1.26);
asparaginase (EC 3.5.1.1); glutamate-oxaloacetate transaminase
(EC 2.6.1.1); glutamin-(asparagin-)ase (EC 3.5.1.38); lysophospho-
lipase (EC 3.1 .1.5); malate dehydrogenase (EC 1.1.1.37);
L
-isoaspartyl/
(
D
-aspartyl)-O-methyltransferase (EC 2.1.1.77); a-sub unit of Glu-
tRNA amidotransferase (EC 6 .3.5 ); isoaspartyl aminopep tidase
(EC 3.4.19.5); amidohydrolase (EC 3.4 , acting on peptide bonds;
EC 3.5 , acting on carbon-nitrogen bonds, other than peptide
bonds).
*These authors contribute d equally to the present work.
(Received 10 May 2004, revised 6 June 2004,
accepted 11 June 2004)
Eur. J. Biochem. 271, 3215–3226 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04254.x
Plant potassium-indep endent asparaginases are evolutio-
narily distinct from bacterial-type asparaginases but show
about 66% s equence similarity to aspartylglucosaminidases
(EC 3 .5.1.26) [26,27]. Although aspartylglucosaminidases
exhibit some asparaginase activity, this is much lower than

their activity towards the natural glycoprotein substrates
[28]. Based on biochemical and crystallographic s tudies
[29–32], aspartylglucosaminidases have been classified as
N-terminal nucle ophile (Ntn) hydrolases [ 33]. In this group
of enzymes, the N-terminal nucleophilic re sidue ( Thr, Se r,
or Cys) is created during an autocatalytic cleavage of the
precursor protein. T here were ear lier suggestions that
asparaginases from higher plants might also belong to the
class of Ntn-hydrolases [ 34], but the experimental e vidence
for this has been very limited [35].
The t wo known Escherichia coli
L
-asparaginases (cytoso-
lic EcAI and periplasmic EcAII) belong to the family of
bacterial-type enzymes. Previously, we have reported [36] o n
the identification and preliminary crystallographic studies
of a potential plant-type asparaginase in this bacterium
(GenBank accession number GI:16128796), termed EcAIII,
which shares a high level of amino acid sequence similarity
(71%) and identity (43%) with the yellow lupin LlA protein
(Fig. 1 ). Originally, the gene encoding EcAIII was
annotated as ybiKandlaterasspt [37,38]. The designations
iaaAandb0828 can also be found in the EcoGene database
( Despite
our original use of the ybiK name [ 36], in the present paper
we favor the iaaA name derived by analogy to a similar
Salmonella gene [39]. This is b ecause although EcAIII and
LlA w ere originally classified as
L
-aspara gina ses, our

present studies indicate that these e nzymes could function
in intracellular degradation of isoAsp-containing proteins.
Modification of
L
-asparagine to isoaspartate is one of the
most common post-translational nonenzymatic covalent
modifications of proteins, usually leading to d egraded func-
tion [40]. There are two ways to inhibit its impact on organ-
isms: repair of the damage or destruction of the modified
proteins. The repair mechanism is based on
L
-isoaspartyl/
(
D
-aspartyl)-O-methyltransferases (EC 2.1.1.77). T hese
enzymes recognize the isoaspartyl residue and in t he
presence of S-adenosylmethionine catalyze ester formation
at the isoaspartyl a-carboxyl group in a methyl transfer
reaction. The e ster converts to succinimide, which after
hydrolysis is converted to
L
-aspartate. Proteins with i soas-
partyl residues can also be degraded by proteolytic e nzymes,
but among the products there will be b-aspartyl (isoAsp)
peptides, for which specific peptidases are needed. One of
these is zinc dipeptidase [41–43] but this enzyme is not able
to hydrolyze all b-aspartyl dipeptides. It is also inactive
towards tripeptides containing b-aspartyl in the first posi-
tion. The degradation of isoAsp peptides is an essential step
Fig. 1. Multiple sequence alignment of aspartylglucosaminidases from Homo sapiens and Flavobacterium meningosepticum and of the present i so-

aspartyl peptidases from Esch erichia c oli (EcAIII) and Lupinus luteus (LlA). The alignment was generated using
CLUSTAL X
version 1.81 [59]. Identical
residues are marked in red, s imilar in yellow. The green arrow indicates the autoproteolytic cleavage site.
3216 D. Borek et al. (Eur. J. Biochem. 271) Ó FEBS 2004
in nitrogen metabolism of some Cyanobacteria, which use
cyanophycin [multi-
L
-arginyl-poly (
L
-aspartic acid) poly-
peptide] as a fluctuating reservoir for the assimilation of
nitrogen [44–51].
Recently, it has been suggested [37] that the E. coli
ybiK g ene product could play a role in the metabolism of
glutathione. The tripeptide glutathione, c-Glu-Cys-Gly, is
widely used to protect cells against o xidative damage
[52] and in Escherichia coli cells can also b e used as an
osmoprotectant [53]. Glutathione synthesis depends on the
glutathione synthase genes ghsAB. However, transport of
exogenous glutathio ne into E. coli has not been character-
ized. A ccording to Parry and Clark [37], expression of the
ybiK gene responded to the presence of cysteine and to
defects in t he cysBgene,andtheybiK knockou t mutation
impaired the use of glutathione as sulfur source. However,
the molecular basis for these observations is not clear. Our
present studies indicate that the amidohydrolase a ctivity
of the ybiK gene product is not directly involved in these
processes.
We describe here our kinetic studies of the isoaspartyl

peptidase and asparaginase activities of the E cAIII Escheri-
chia coli iaaA gene product and of the plant homolog LlA
from Lupinus luteus. Our findings, combined with previous
studies, demonstrate that hydrolysis of isoAsp peptides is
the dominant a ctivity of t hese enzymes, and s uggest at the
same time a very b road role for po tassium-independent
asparaginases in plants.
Materials and methods
Bacterial strains, plasmids, and media
The sequence encoding LlA was obtained from a cDNA
library from yellow lupin roots infected with Bradyrhizo-
bium sp. [54]. E. coli strain DH5a genomic DNA was used
as template in a PCR to obtain the sequence encoding
EcAIII. For subcloning and manipulation, th e E. coli
DH5a strain was used as host in both cases. Bacteria were
grown at 37 °C in Luria–Bertani broth, Lennox formula-
tion (LB) for small-volume cultures or in medium contain-
ing 1% (w/v) tryptone, 0.4% ( w/v) NaCl and 0 .5% (w/v)
glucose for large-volume cultures. When required, either
ampicillin or chloramphenicol was added at a concentration
of 100 or 25 lgÆmL
)1
, respectively.
Cloning of LlA and EcAIII sequences
DNA manipulations were performed using standard tech-
niques [55]. The LlA construct was obtained and cloned into
pET-15b (Novagene, Madison, W I, USA) as described
previously [22]. This plasmid was used to transform E. coli
BL21-CodonPlusÒ (Stratagene, La Jolla, CA, USA). To
prepare the DNA fragment for the EcAIII coding sequence,

primers complementary to each end of the ORF were
synthesized (MWG Biotech, Ebersberg, Germany) as
follows: EcoNase1 5 ¢-GACGAATACC
ATGGGCAAA
GCAGTC-3¢ and EcoNase2 5¢-ACATTACCGGATC
CAAGTTCACTGTGTGGC-3¢. These primers introduce
restriction sites for NcoI (incorporating the initiator codon,
underlined) and BamHI, respectively. Standard PCR
amplification was performed. The target fragment was
digested with NcoI+BamHI (N ew England B iolabs,
Beverly, MA, USA), purified from agarose gel (Qiagen,
Hilden, Germany), ligated into the pET-11d vector (Nova-
gene), and transformed into E. coli JM109 cells. T he
resulting recombinant p lasmid was then used to transform
the E. coli strain BL21(DE3)pLysS (Novagene).
Expression and purification of LlA
Cultures (1 L) in the exponential growth phase (D ¼ 0.7 at
600 n m) were induced with 1 m
M
isopropyl thio-b-
D
-galactoside (IPTG) and shaken for 4 h at 300 r.p.m. at
37 °C. Cells were centrifuged at 4000 g,4 °C for 15 min and
lysed in 100 mL of a solution containing 30 m
M
Tris/HCl,
pH 8.0, 1 m
M
phenylmethylsulfonyl fluoride, and 15 m
M

2-mercaptoethanol. T he lysate was s onicated and centri-
fuged at 4000 g,4 °C for 30 min. MgCl
2
solution was added
to the supernatant to a final concentration of 50 m
M
and the
mixture was stirred for 30 min at 4 °C to remove DNA. The
solution was centrifuged at 6500 g,4 °Cfor30 minat4°C.
The supernatant was dialyzed against binding buffer
containing 5 m
M
imidazole, 0.5 m
M
NaCl and 20 m
M
Tris/HCl, pH 7.9, and then applied to the HiTrap column
prepared as described previously [22]. T he column was
equilibrated with b inding buffer and sub sequently wash
buffer (30 m
M
imidazole, 0.5 m
M
NaCl, and 20 m
M
Tris/
HCl, pH 7.9) was applied to remove nonspecifi cally bound
proteins. The expected product was eluted with a buffer
containing 1
M

imidazole, 0.5 m
M
NaCl, a nd 20 m
M
Tris/
HCl, pH 7.9. The p rotein-containing fractions were con-
centrated to 4 mL volume and buffer was exchanged to
20 m
M
Tris/HCl, pH 8.5, 0.1
M
NaCl, and 10% glycerol
using Centricon-YM-30 filters ( Millipore, Billerica, MA,
USA). The sample was a pplied to a Superdex 75 HiLoad
16/60 gel filtration column (Amersham Bioscience AB,
Uppsala, Sweden) equilibrated with the same buffer. The
product was eluted in one peak corresponding to a
molecularmassof% 75 kDa. The product-containing
fractions were concentrated as described above. The sample
purity was analyzed by SDS/PAGE.
Expression and purification of EcAIII
Cultures (1 L) in the exponential phase of growth (D ¼ 0.6
at 600 nm) were induced with 1 m
M
IPTG and shaken for
3 h at 300 r.p.m. at 37 °C. Cells were centrifuged at 4000 g,
4 °C for 30 min and lysed at 4 °C in 100 mL of a solution
containing 30 m
M
Tris/HCl, pH 8.0, 10 m

M
EDTA, 1 m
M
dithiothreitol, 0 .25 m
M
phenylmethylsulfonyl fluoride, and
lysozyme (40 lgÆmL
)1
). The lysate was frozen on dry ice
and thawed 3–4 times at room temperature. It was then
centrifuged at 5000 g,4°Cfor1h.MgCl
2
solution was
added t o the supernatant to 25 m
M
concentration a nd the
mixture was stirred for 1 h at 4 °C to remove DNA. Finally,
the solution was centrifuged at 4000 g for 1 h a t 4 °C. The
protein was purified in a three-step procedure including:
fractionation with poly(ethylene glycol) (PEG8K), anion-
exchange chromatography, and gel fi ltration. The purifica-
tion procedure was carried out at 4 °C, except for the gel
filtration step, which was carried out at 4 °Cforsome
batches, and a t room temperature for others. The superna-
tant was stirred for 1 h after addition of solid PEG8K to
Ó FEBS 2004 Homologous L. luteus and E. coli amidohydrolases (Eur. J. Biochem. 271) 3217
9% concentration. The suspension was centrifuged a t
4000 g,4°C for 30 m in and the pellet was removed. To
the supernatant, further PEG8K was added to a concen-
tration of 35% and stirring continued for 1 h . The solution

was then centrifuged as before. The supernatant was
discarded and the pellet was dissolved in buffer A contain-
ing 100 m
M
Tris/HCl, pH 8.5, 150 m
M
NaCl, and 5%
glycerol, and then dialyzed against the same buffer. The
dialyzed protein solution was centrifuged a t 8500 g,4 °Cfor
30 min and the supernatant w as applied to manually
prepared DEAE-cellulose ion-exc hange column or com-
mercially available Mono-Q column (MonoQ HR 10/10,
Amersham Bioscience AB) equilibrated with buffe r A. After
washing with buffer A, the protein was eluted as a single
peak (at 350 m
M
NaCl) by application of a 150–500 m
M
linear NaCl g radient in buffer A . The
L
-asparaginase
activity was checked using Nessler reagent to detect
ammonia release. P rotein-containing f ractions were dia-
lyzed against buffer A and concentrated to 2 mL volume
using Centricon-YM-30 filters (Millipore). The sample was
applied to a gel-filtration c olumn (S300 H S ephacryl,
Amersham Bioscience AB, Uppsala, Sweden) e quilibrated
with buffer A and t he protein w as eluted in one peak
corresponding to a molecular mass of % 66 kDa. Active
fractions were concentrated as described above. The purified

enzyme was a nalyzed by SDS/PAGE a nd purity w as
estimated visually to be higher than 95%.
Enzymatic assays and determination of kinetic
parameters
The following substrates: GlcNAc-
L
-Asn, b-
L
-Asp-
L
-Leu,
Gly-
L
-Asn,
L
-Gln,
L
-Asn a-amide,
L
-Asp a-amide, and
L
-Asn (Sigma) were used to assay the activity of the LlA and
EcAIII proteins. With some of the substrates, no enzymatic
activity could be detected, indicating lack or possibly very
low level of the co rresponding activity. Enzyme activity for
reactions where
L
-aspartate was one of the products was
assayed by a coupled enzymatic procedure [56] based on (a)
the hydrolysis reaction releasing the aspartate; (b) subse-

quent transamination of the aspartate to oxaloacetate by
glutamate-oxaloacetate t ransaminase (GOT) in the presence
of a-ketoglutarate, and (c) formation of NAD
+
after
malate dehydrogenase (MDH)-mediated reduction of
oxaloacetate to m alate with NADH as cofactor. The
decrease of NADH concentration was measured spectro-
photometrically using a Hewlett-Packard 8452 A spectro-
photometer at a wavelength of 360 nm. All reagents and
enzymes for the GOT and MDH steps were obtained from
Sigma. Each reaction was performed in 1 m L of 20 m
M
Tris/HCl, pH 7.5, containing 0.1 m
M
a-ket oglutarate,
0.2 m g of NADH, 12.5 units each of GOT a nd MDH, as
well as a substrate and an appropriate amount of the
enzyme. After the assay mixture had been incubated for
about 10 min at room temperature, the enzyme was added
and timed measurements of the initial rates were performed.
Preliminary controls, using reaction mixtures that contained
L
-Asn but not the enzymes of interest, revealed a decrease of
NADH concentration due to aspartate contamination of
the commercial
L
-Asn samples. Thus, it was essential to
perform for each reaction a blank test without added
enzyme. The differences between the measurements with

and without EcaIII or LlA were calculated and these
background-corrected values were used in the subsequent
stages of the calculations. T o determine K
m
and k
cat
values,
7–10 different concentrations of a substrate were used,
generally ranging from % 0.3–10· the K
m
.Whenaspartate
was not the product, we used the Nessler reagent (Aldrich)
to measure the release o f ammonia [57]. This colorimetric
method utilizes alkaline (KOH) solution of an iodide
complex o f mercury (II). Measurements were performed
spectrophotometrically at 414 nm w avelength. Calibration
curves were prepared using known concentrations of
ammonium sulfate. Each reaction was performed using
100 lL o f t he enzyme solution mixed with 900 lLofa
solution with a given concentration of a substrate in 20 m
M
Tris/HCl, pH 8.5. The mixture was incubated at 37 °Cand
every 10 min 50 lL were transferred to separate 1 mL
cuvettes containing 0.1 mL of 15% trichloroacetic acid
solution. After the reaction was quenched, 0.65 mL of
Nessler reagent dissolved in H
2
O (1 : 6.5, v/v) were added.
Spectrophotometric measurements were performed after
incubation for 15 min at room temperature.

Electrospray-ionization mass spectrometry
Trypsin digestion of 1 lg o f EcAIII was carried out in
25 m
M
NH
4
HCO
3
for 12 h at 37 °C using 12.5 ng lL
)1
of
trypsin. The tryptic peptides were reduced with 10 m
M
dithiothreitol for 30 min and alkylated with 55 m
M
iodo-
acetamide for 30 min at room temperature. The sample was
diluted in 0.1% trifluoroacetic acid and applied t o a reverse-
phase HPLC 300 lm · 5 mm C18 precolumn (LC Pac-
kings, Sunnyvale, CA) with 0.1% trifluoroacetic acid as the
mobile phase. The eluate was subjected to a 75 lm · 15 cm
C18 column (LC Packings) and peptide s eparation was
carried out at 0.2 lLÆmin
)1
with a linear gradient o f
acetonitrile from 0–25% (v/v) in 25 min in the p resence of
0.05% formic acid. The column outlet was coupled to a
Q-TOF electrospray mass s pectrometer (Micromass). Mole-
cular mass analysis w as performed using the nano-Z-spray
ion source of the spectrometer working in the regime of

data-dependent MS to MS/MS switch, allowing for a 3 s
sequencing scan for each detected double- and triple-charge
peptide. The data w ere analyzed using the
MASCOT
program
(). Additionally, M S meas-
urements of intact EcAIII and LlA were performed. Protein
solution (1 lgÆmL
)1
) in 0.05% formic acid was injected at 5
lLÆmin
)1
flow rate to the micro-Z-spray ion source of t he
mass spectrometer. For EcAIII, the MS mass measurements
were repeated for p rotein recovered from c rystals that had
been stored for over one year. The data were analyzed using
the
MASSLYNX
software (Micromass).
Thermostability studies
Thermal denaturations were performed on a J-715 spectro-
polarimeter (Jasco, Tokyo, Japan) following the ellipticity at
222 n m at 2 nm bandwidth and response time o f 4 s in
25 m
M
Tris/HCl, 300 m
M
NaCl,pH7.5,at1°CÆmin
)1
heating rate. Protein concentration was 100 lgÆmL

)1
.
Analysis of the data was performed by the
PEAKFIT
software
(Jandel Scientific Software, San Rafael, CA). Additionally,
the effect on enzymatic activity was asse ssed u sing the
3218 D. Borek et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Nessler assay. The tested enzyme was preincubated with
L
-Asn at different temperatures at 5 °C intervals, after
which the r eaction was stopped a nd the a ctivity was
measured as described above.
Sequence analysis
Amino a cid sequences were retrieved from the Swiss-Prot/
TrEMBL databases [58]. M ultiple sequence alignments for
76 sequences were performed with
CLUSTAL X
Version 1.81
[59]. The phylogeny was inferred from 423 amino acid sites.
Phylogenetic trees were calculated u sing the neighbor-
joining method with correction for multiple substitutions.
The bootstrap trees were calculated with 1000 bootstrap
trials. Maximum likelihood tree distances were compu-
ted with
TREE
-
PUZZLE
5.0 [60] using t he Jones–Taylor–
Thornton substitution matrix [61] and amino acid frequen-

cies observed in the sequences under analysis. Site-to-site
rate variation was modeled u sing a gamma distribution w ith
eight gamma rate categories. The above settings and the
shape of the gamma distribution a-parameter estimated
from the alignment (1.03 ± 0.07) were used for the
bootstrap a nalysis. The maximum likelihood tr ees w ere
generated by the quartet puzzling procedure with 10 000
puzzling steps and the program settings described ab ove.
Results
Expression and purification
LlA. LlA was purified to a high level of homogeneity,
allowing use of the protein for biochemical and crystallo-
graphic studies. Yields were 6 mg of pure protein per litre of
culture, eluted from the final size exclusion chromatography
column in a single peak corresponding to a molecular mass
of % 75 kDa. The purity of the protein was checked by
visual inspection of SDS/PAGE g els. It was observed that
on maturation, which was complete in less than 3 days at
4 °C, the protein underwen t an a utocatalytic cleavage
characteristic of Ntn-hydrolases [62–64], leading to the
release of t wo subunits, of 23 kDa (a-subunit) and 14 k Da
(b-subunit) (Fig. 2A). The N-terminal His-tag, present in
the recombinant protein to facilitate the purification
process, was not removed from the final product because
it did not deactivate the enzyme.
EcAIII. The expression and purification protocol of EcA-
III yielded 30 mg of pure protein per 1 L of culture. The
protein was purified to homogen eity (Table 1) sufficient for
biochemical a nd cr ystallographic studies [36]. E ven at
intermediate purification steps, complete au toproteolytic

maturation was observed leading to two s ubunits with
approximate masses o f 19 k Da (a-subunit) and 14 k Da
(b-subunit) (Fig. 2B). The maturation process was faster at
room temperature and slower at 4 °C, but could not be
avoided even when the entire purification procedure was
performed very quickly at 4 °C (Materials and methods).
Moreover, an additional protein band with molecular mass
% 17 kDa was observed on the SDS/PAGE gels, indicating
that the a-subunit undergoes further autoproteolysis. It was
observed that after 48 h of incubation at 4 °C, the a-subunit
was fully converted to the shorter variant. Some preliminary
experiments indicate that this secondary cleavage process
can be i nhibited by Zn
2+
ions. Other divalent cations have
no effect on this process (Fig. 3).
Mass spectrometry
LlA. The mass spectrum of the intact mature protein
contains two prominent peaks, corresponding to polypep-
tide chains with molecular masses of 22893 and 13605 Da.
This confirms an autocleavage process resembling the
maturation process of aspartylglucosaminidases. Base d on
the LlA sequence, the 22893 Da peak can be assigned to
the N -terminal s ubunit a, including residues up to Gly192
of the precursor sequence, extended at the N-terminus
by the 19 amino acids of the His-tag sequence
(GSSHHHHHHSSGLVPRGSH-) with a molecular mass
of 2032 Da. Originally, this additional sequence, introduced
by the pET-15b vector, contained an N-terminal methionine
residue, but this methionine is removed in the expression

system by a bacterial methionyl aminopeptidase [65]. The
peak at 13605 Da corresponds exactly to the b subunit,
comprising residues Thr193–Thr325 of the C-terminal part
of the precursor.
EcAIII. The mass spectrum of the mature protein contains
two prominent peaks, corresponding to molecular masses
Fig. 2. SDS/PAGE analysis of the progress of purification of LlA and
EcAIII. (A) LlA: M, molecular m ass marker; N, after affinity chro-
matography; GF, fractions after gel filtration; (B) EcAIII: M,
molecular mass marker; MQ, after ion exchange chromatography;
GF, after gel filtration.
Ó FEBS 2004 Homologous L. luteus and E. coli amidohydrolases (Eur. J. Biochem. 271) 3219
of 17091 D a a nd 13852 Da, which can be assigned to t he
a-andb-subunits, respectively. The positions of these peaks
are exactly the s ame for the protein recovered from crystals.
However, the molecular masses for these two subunits
predicted from the amino acid sequence o n the assumption
of autocat alytic c leavage at Gly178–Thr179 (Fig. 1) are
18993 and 1 4419 Da, respectively. This suggests that after
the activating event of proteolytic cleavage, t he p rotein
undergoes f urther proteolysis producing a-andb-subunits
that are shorter than expected. As we were unable to assign
the observed masses to any particular amino acid sequences,
an additional MS/MS sequencing experiment was per-
formed. The combination of tandem M S and overall mass
measurement allowed us t o assign the 13852 Da peak to the
b-subun it composed of residues Thr179–Gly315 and con-
taining three oxidized methionine residues, as suggested by
MS/MS. The MS/MS sequencing also confirms the absence
of the N-terminal Met1 residue of the a-subunit, in

agreement with the predicted cleavage of the Met1-Gly2
sequence by the bacterial methionyl aminopeptidase [65].
However, even taking this into account, the length of the
a-subun it is not immediately obvious. The last residue
detected by the M S/MS sequencing is A rg157, but the
measured overall mass is higher t han for a polypeptide
chain terminating with th is residue. This suggests that there
are a few additional amino acid s at t he C-terminus of the
a-subun it, which could not be detected by MS/MS
sequencing due to insufficient length of the remaining
peptides. The closest match is obtained f or Ala161. The
molecular mass of 17091 Da found in the MS spectrum c an
be explained by the Gly2–Ala161 polypeptide exactly if one
assumes (in agreement with MS/MS) that one of the
methionine residues is oxidized and that the sodium cation,
found by crystallographic s tudies to be tightly c oordinated
by the a-subunit (D. Borek & M. Jaskolski, unpublished
results), also contributes to the total mass.
Enzyme assay and determination of kinetic parameters
A s ummary of the kinetic data is presented in Table 2. The
protein concentration of both enzymes was d etermined
using the Bradford metho d [66]. For EcAIII, the calculation
of k
cat
was based on molar concentratio n determined for the
shorter versions of both subunits, as obtained from mass
spectrometry.
Both enzymes could hydrolyze
L
-asparagine and a

b-p eptide formed through t he Asp side c hain. B locking of
the a-amino group (as i n Gly-
L
-Asn)orofthea-carboxyl
group (as in
L
-asparagine a-amide) of
L
-asparagine resulted
in inactive substrates (Table 2). The latter compound,
together with
L
-glutamine, which is also inactive, demon-
strates in addition that the hydrolyzed amide function
cannot be either a-orc-, but must be precisely b The
enzymes are also unable to hydrolyze b-N-glycosylated
L
-asparagine side chains, and therefore have no aspartyl-
glucosaminidase activity.
Table 1. Purification progress for EcAIII.
Measure
Crude
extract
Solution after
poly(ethylene glycol)
precipitation
Ion exchange
(pool and
concentrated)
Gel filtration

(pool and
concentrated)
Volume (mL) 198.0 95.0 37.1 1.2
C
protein
(mgÆmL
)1
) 42.1 8.0 4.2 12.3
Asparaginase activity (UÆmL
)1
) 9.2 17.6 20.4 355.8
Total protein (mg) 8336 760 156 14.8
Total asparaginase (U) 1822 1672 757 427
Specific activity (UÆmg
)1
) 0.22 2.2 4.8 28.8
Yield per step (%) 100 91.8 45.3 56.4
Total yield (%) 100 91.8 41.5 23.4
Total fold purification 1 10.0 21.7 131
Fig. 3. SDS/PAGE analysis of the secondary c leavage of EcAIII. (A)
Incubation at room te mperature for 2 h: M, molecular mass marker;
NM, 3 lgÆlL
)1
protein in 20 m
M
Tris/HCl,pH8.5,withoutanymetal
cations; Mg, 1 0 lLNM+10lL10m
M
MgCl
2

;Ca,10lL
NM + 10 lL10m
M
CaCl
2
;Zn,10lLNM+10lL10m
M
ZnCl
2
,
Li, 10 lLNM+10lL10m
M
Li
2
SO
4
. (B) As above, but with
incubation time of 48 h. The protein band s are labeled a s follows:
pro-a, i ntact s u bunit a (19 kDa); a, shortened subunit a (17 kDa);
b, subunit b (14 kDa).
3220 D. Borek et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Thermostability studies
Aspartylglucosaminidase from Homo sapiens is thermo-
stable and retains activity up to 80 °C [67]. In contrast,
measurements for the present e nzymes indicate lower
thermal stability. The melting temperature for EcAIII
obtained b y monitoring a CD signal at 222 nm was
59.2 °C. Nessler activity assays are i n agreement with t his
value and indicate loss of enzymatic activity at 60 °Cfor
both proteins.

Sequence analysis
The phylogenetic analysis was carried out for 76 amino acid
sequences related t o EcAIII and LlA retrieved from the
Swiss-Prot/TrEMBL database. The neighbor-joining (N-J)
tree of these sequences has four major branches. Because it
was too complicated for graphical display, a simplified
version with 42 representative sequences was also calculated.
The sequences were selected in such a way that the overall
topology of t he tree remained unchanged (Fig. 4 ). Similar
topologies were obtained using the maximum likelihood
(ML) method, but the resolution of the trees was too low for
separation of some branches. One of the branches comprises
mainly archaeal enzymes, another one contains eukaryotic
and bacterial aspartylglucosaminidases, and the third group
contains plant a nd bacterial enzymes with isoas partyl
peptidase a ctivity. A small fourth branch is established
by eukaryotic enzymes with unknown biochemical chara-
cteristics. In both the ML and N-J trees the b ranch
corresponding to aspartylglucosaminidases is clearly dis-
tinguishable, but for the ML trees the resolution of the
archeal and plant/bacterial branches makes a separation
difficult.
The archaeal branch s plits into two distinct clusters, but
the branching of the sequences is not congruent with 16S
rRNA phylogeny. One branch is shared by Crenarchaeota
(Sulfolobus and Pyrobaculum species) and Euryarchaeota
(Pyrococcus species). The second one is composed of
Euryarchaeota (Thermoplasma spp.) and Cyanobacteria.
The presence of bacterial sequences in this cluster indicates
that lateral gene transfer has played an important role

between the Archaea and Cyanobacteria. The branch of
aspartylglucosaminidases is clearly separated and c onsists of
two groups: b acterial and eukaryotic sequences. In contrast
to the archaeal branch, there is congruency with 16S rRNA
phylogeny for t he eukaryotic sequences. The third b ranch,
annotated as Ôisoaspartyl peptidasesÕ in Fig. 4, is divided
Table 2. Summary of kinetic parameters characterizing the enzymatic activities of EcAIII and LlA with respect to various substrates.
Ó FEBS 2004 Homologous L. luteus and E. coli amidohydrolases (Eur. J. Biochem. 271) 3221
into two clusters: one corresponding mostly to plant
enzymes a nd the other to bacterial enzymes. The sequences
in the plant group are closely related.
Discussion
In this paper, we have des cribed p rocedures for successful
expression andpurification of twoamidohydrolytic enzymes,
from Escherichia coli (EcAIII) and from Lupinus luteus
(LlA), as well as their biochemical characterization. The
expression levels in E. coli cells were high and yielded 6 (LlA)
or 30 mgÆL
)1
(EcAIII) of very pure protein. Purification of
EcAIII required a three-step procedure while introduction of
a His-tag sequence to L lA reduced the purification procedure
to two steps. The His-tag was not removed from the purified
LlA p rotein, as it did not appear to affect the e nzyme a ctivity.
Fig. 4. Bootstrap tree of amino acid sequences of Ntn-amidohydrolases. The tree w as calculated from th e
CLUSTAL X
version 1.81 alignment [59].
Bootstrap valu es h igh er t han 50% are included. Acc ession numbers of se quences are in parentheses. Red branch, m ostly a rcheal asparaginases; blue
branch, eukaryotic and bacterial aspartylglucosaminidases; green branch, plant-type asparaginases and their bacterial homologues; brown branch,
eukaryotic sequenc es with unkn own bioche mical chara cteristics. The sequ ences of the enzymes studied in this work (L lA and E cAIII) ha ve bee n

underlined.
3222 D. Borek et al. (Eur. J. Biochem. 271) Ó FEBS 2004
The full sequences of both protein constructs for e xpres-
sion have an N-terminal methionine residue. However, in
each case this Met residues is followed by a glycine: Gly2 of
the His-tag sequence in the case of LlA, and the Gly2
residue of the native EcAIII sequence. As in E. coli there is a
methionyl aminopeptidase w hich removes N-terminal Met
residues with efficiency that is inversely proportional to the
size of the following amino acid [65], both purified proteins
lack the first methionine residue. In EcAIII this lack of Met1
seems t o be a natural feature but in the case of LlA there is
still a non-native N-terminal sequence introduced by the
pET-15b vector, so the absence of the initial methionine is in
this case less important. However, it is interesting to note
thatthenativesequenceofLlAbeginswithMet-Gly-,
exactly as in EcAIII (Fig. 1).
Both enzymes undergo a utoproteolysis, which can be
detected by SDS/PAGE, and form mature heterotetramers
(ab)
2
, a s deduced from gel filtration chromatography. The
maturation process itself seems to vary slightly, because for
EcAIII, after the primary cleavage event, further proteolytic
trimming at the C-termini of both subunits takes place,
which, however, has no impact on the enzyme’s activity. No
such trimming can be detected for LlA. In particular, the
original subu nit a of EcAIII (19 kDa) is converted on
incubation to a shorter variant ( 17 kDa) (Fig. 3 ). This
pattern of maturation o f EcAIII, which makes it similar to

human aspartylglucosaminidase [68], was confirmed for
material recovered from dissolved crystals, w here only the
shorter version of subunit a could be detected (data not
shown). As the EcAIII crystals were grown very quickly in
the presence of MgCl
2
[36], we speculated that the cleavage
could be promoted by magnesium. This prompted a series
of incubation experiments with different metal cations
which revealed that the presence of metals had no effect on
the maturation process, except for ZnCl
2
, which stopped the
truncation of subunit a completely (Fig. 3 ). As the two salts,
MgCl
2
and Z nCl
2
, share the same anion, one can identify
the zinc cation as the inhibitor. Additionally, this result
indicates t hat the process of further cleavage is not an
artefact of contamination by metalloproteases, whic h typi-
cally are zinc-dependent. A speculative hypothesis about the
role of zinc is based on the observation that the short spacer
at the C-terminus of subunit a includes a His r esidue
(Fig. 1 ), a preferred ligand for Zn
2+
coordination. Zinc
binding by the spacer sequence could change its conforma-
tion and make it unavailable for the second s tep of

maturation.
Tandem mass spectrometry for EcAIII proves that the
first residue of the b subunit is a threonine (Thr179), as
predicted f rom s equence alignments (Fig. 1) with structur-
ally characterized aspartylglucosaminidases. This result
allows the identification of Thr179 (and its T hr193 analog
in LlA) as the catalytic nucleophile and the classification of
both enzymes as Ntn-hydrolases.
The kinetic experiments demonstrate that LlA and
EcAIII have both
L
-asparaginase as well as isoaspartyl
peptidase activities. The affinity for b-
L
-Asp-
L
-Leu is over
one order of magnitude higher than that for L-Asn, and the
specificity index k
cat
/K
m
almost two orders of magnitude
higher, for both enzymes. These findings are somewhat in
contrast with an earlier report on Arabidopsis thaliana
asparaginase, which found that it had comparable affinities
for b-
L
-Asp-
L

-Leu and
L
-Asn [35]. Our results suggest that
these enzymes serve primarily as isoaspartyl peptidases and
that their
L
-asparaginase activity is of secondary importance
although it may bring additional benefits for the organisms.
Modification of asparagine residues to isoaspartyl pep-
tides is the most common modification in mature proteins.
It is also one of the most dangerous modifications, as it
causes a structural change that may significantly alter a
protein’s three-dimensional structure, leading to a loss or
change of activity, degradation, or aggregation [69,70]. A
repair mechanism exists that involves
L
-isoaspartyl/
(
D
-aspartyl)-O-methyltransferase (EC 2.1.1.77) action in
the presence of S-adenosylmethionine as a methyl donor
to convert isoaspartyl to aspartyl residues. However, this
methyltransferase activity is highly dependent on local
sequence around the isoaspartyl modification [71,72], with
preferences against n egatively charged side chains close to
the carboxyl part of the isoaspartyl residue. Moreover, the
site of the isoaspartyl modification has to be accessible for
the r epair r eaction. In situations where the modification
cannot be repaired, the damaged protein should be d egra-
ded. Among the proteolytic products there will be dipep-

tides containing N-terminal isoaspartyl residues. a-Peptide
bond specific peptidases cannot recognize peptide bonds
formed by side chains, and thus are not able to degrade
b-aspartyl peptides, which require specialized hydrolytic
enzymes. It has b een reported that in E. coli the product of
the iadA g ene is a zinc isoaspartyldipeptidase [41,42].
However, this enzyme cannot hyd rolyze some of the
b-aspartyl dipeptides, and its affinity for b-
L
-Asp-
L
-Leu
is relatively low (K
m
¼ 0.8 m
M
). Furthermore, E. col i
mutants deficient i n the iadA gene still retain the ability to
hydrolyze b-aspartyl dipeptides [41]. It is likely that EcAIII,
the product of the E. coli iaaA gene, and its plant analogues
represented by LlA, a re the m issing link o f this m etabolic
pathway. The lack of activity for Gly-
L
-Asn in dicates t hat
the enzymes are aminopeptidases. In aspartylglucosamini-
dases, a f re e a-amino group is not required for enzyme
activity and can be substituted by a group or an atom with
comparable size [73]. It would t hus appear that discrimin-
ation at the a-amino position of the substrates is more
connected with the size of t he substituent than with a

specific pattern of interactions.
The f act that
L
-Asn amide is not recognized indicates t hat
the specificity of EcAIII and LlA, as well as of aspartylglu-
cosaminidases [73], is limited to substrates which possess
afreea-carboxyl group. Additionally, as no activity for
L
-Asp/
L
-Asn a-amides was detected, it is clear that only
amides in the b-position can be hydrolyzed. In other words,
an alternative docking mode of a substrate amino acid, with
the a-andb-amide groups interchanged, does not lead to
productive catalysis. This, together with the observation of
theroleofthea-amino substituent [73], might suggest that it
can be accommodated in only o ne way, directing t he correct
orientation of a substrate in the active site. The length of the
linker presenting the amide group for hydrolysis i s also
important, because
L
-Gln is not hydrolyze d, although its
both a functions are perfectly acceptable.
The fact that these two enzymes do not show any
aspartylglucosaminidase activity might be somewhat sur-
prising in view of the rather considerable sequence similarity
(Fig. 1 ). However, detailed phylogenetic analysis reveals
Ó FEBS 2004 Homologous L. luteus and E. coli amidohydrolases (Eur. J. Biochem. 271) 3223
that enzymes with aspartylglucosaminidase activity and
the present plant-type amidohydrolases belong to different

branches, suggesting that the plant-type enzymes do have
their idiosyncratic features which must be reflected in the
architecture of the active sites. O bviously, EcAIII and LlA
have a more restricted substrate spectrum than aspartylglu-
cosaminidase, which are also able to hydrolyze b-aspartyl
peptides [74]. The plant-type enzymes also have lower
thermostability than aspartylglucosaminidases, and do not
share the latter’s SDS resistance (data not shown).
The question arises why LlA and EcAIII would be
endowed with dual activity. Previous studies [22] have
shown that LlA and its close homologs from d ifferent
Lupinus species really serve as asparaginases in develo ping
seeds [26,27]. A possible explanation of the other, i soas-
partyl peptidase, ac tivity is that the seeds have to retain
their a bility t o grow for a very long time. During t he
storage period, their proteins c an undergo modification
and isoaspartyl peptidase activity is necessary to destroy
the altered proteins and t o allow only the healthy seeds to
grow. The presence of
L
-asparaginase activity agrees also
with the usage of
L
-asparagine, the ma in storage com-
pound, as a n itrogen source for protein syn thesis. The fact
that the asparaginase activity decreases after the assimil-
ation of atmospheric nitrogen has started [26], confirms its
role in managing the nitrogen reservoirs. It is an elegant
analogy to the role of the homologous enzymes from
Cyanobacteria in managing the cyanophycin supply, which

is considered to be a d ynamic reservoir of n itrogen for
Cyanobacteria [51].
The true role of EcAIII remains unclear, however. Its K
m
for
L
-asparagine is comparable to that of t he cytosolic
bacterial-type asparaginase, EcAI ( 3.5 m
M
) but is much
higher than the K
lÀAsn
m
value for the periplasmic enzyme,
EcAII (11.5 l
M
) [75]. Su ch a low affinity of EcAIII and the
presence of another enzyme with m uch higher affinity for
L
-asparagine a rgue against the hypothesis t hat the enzyme
may serve as an asparaginase. Regarding the isoaspartyl
peptidase activity of EcAIII the situation is clearer, but still
not without open questions. It is known for example that
bacteria with zinc isoaspartyl dipeptidase gene dysfu nction
may survive due to the iaa A gene product act ivity, but the
behavior of an organism with an iaaA knockout has not
been investigated.
Recent studies have suggested that enzymes like L lA
and EcAIII might be involved not only in c arbon and
nitrogen but also sulfur metabolism [ 37]. Glutathione

(c-Glu-Cys-Gly) catabolism largely concerns the remobi-
lization o f cysteine, for example for protein synthe sis
during s eed storage and dur ing sulfur deprivation. The
studies of Parry and Clark suggest that t he iaa Agene
product in E. coli could be involved in glutathione
transport, a s a cysB/iaaA double mutant grows only
weakly with glutathione as the sole source of sulfur [37].
However, in view of the present results, it seems unlikely
that the iaaA gene product could be involved directly in
glutathione catabolism, namely in the hydrolysis of t he
c-Glu-Cys dipeptide, as the e nzyme lacks glutaminase
activity. This feature also distinguishes EcaIII from
bacterial-type asparaginases, which can hydrolyse
L
-Gln
as well. The elucidation of the true physiological role of
EcAIII clearly requires further studies.
Acknowledgements
We wish to thank Prof. Michal Dadlez, Jacek Oledzki, and Jacek
Sikora (Institute of Biochemistry and Biophysics, Polish Academy of
Sciences, Warsaw) for their help and discussion of the mass spectrom-
etry results. This work was supported in p art by a subsidy from the
Foundation for Polish Science to M. J.
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Supplementary material
The f ollowing material is available from http://blackwell
publishing.com/products/journals/suppmat/EJB/EJB4254/
EJB4254sm.htm
Fig. S1. Kinetic analysis of isoaspartyl peptidase activity of
(a) EcAIII and (b) LlA.
3226 D. Borek et al. (Eur. J. Biochem. 271) Ó FEBS 2004