Mickiewicz University, Poznan, Poland;2Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland;3Molecular Medicine Unit, Un
Trang 1Expression, purification and catalytic activity of Lupinus luteus
Dominika Borek1,*, Karolina Michalska1,*, Krzysztof Brzezinski1, Agnieszka Kisiel2, Jan Podkowinski2, David T Bonthron3, Daniel Krowarsch4, Jacek Otlewski4and Mariusz Jaskolski1,2
1
Department of Crystallography, Faculty of Chemistry, A Mickiewicz University, Poznan, Poland;2Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland;3Molecular Medicine Unit, University of Leeds, UK;4Laboratory 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)1of culture, respectively The enzymes
are heat-stable up to 60C and show both isoaspartyl
di-peptidase andL-asparaginase activities Kinetic parameters
for both enzymatic reactions have been determined, showing
that the isoaspartyl peptidase activity is the dominating one
Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected 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 consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine 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 ammonia
Using amino acid sequences and biochemical properties as
criteria, enzymes with asparaginase activity can be divided
into several families [1] The two largest and
best-charac-terized families 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 of some types of lymphoblastic leukemias [2–6] Their homologues are 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 is necessary for protein synthesis There are two groups of such proteins, called potassium-dependent and potassium-independent aspara-ginases We have reported previously the identification and sequencing of cDNA (GenBank accession number GI:4139265) coding for a protein (termed LlA) from yellow lupin (Lupinus luteus) that belongs to the 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 the 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 nitrogen 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: mariuszj@amu.edu.pl
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: N4-(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-subunit of
Glu-tRNA amidotransferase (EC 6.3.5.-); isoaspartyl aminopeptidase
(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 contributed equally to the present work.
(Received 10 May 2004, revised 6 June 2004,
accepted 11 June 2004)
Trang 2Plant potassium-independent asparaginases are
evolutio-narily distinct from bacterial-type asparaginases but show
about 66% sequence 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 studies
[29–32], aspartylglucosaminidases have been classified as
N-terminal nucleophile (Ntn) hydrolases [33] In this group
of enzymes, the N-terminal nucleophilic residue (Thr, Ser,
or Cys) is created during an autocatalytic cleavage of the
precursor protein There were earlier suggestions that
asparaginases from higher plants might also belong to the
class of Ntn-hydrolases [34], but the experimental evidence
for this has been very limited [35]
The two known Escherichia coliL-asparaginases
(cytoso-lic EcAI and periplasmic EcAII) belong to the family of
bacterial-type enzymes Previously, we have reported [36] on
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 ybiK and later as spt [37,38] The designations
iaaA and b0828 can also be found in the EcoGene database
(http://bmb.med.miami.edu/EcoGene/EcoWeb/) Despite our original use of the ybiK name [36], in the present paper
we favor the iaaA name derived by analogy to a similar Salmonellagene [39] This is because although EcAIII and LlA were originally classified as L-asparaginases, our present studies indicate that these enzymes could function
in intracellular degradation of isoAsp-containing proteins Modification ofL-asparagine to isoaspartate is one of the most common post-translational nonenzymatic covalent modifications of proteins, usually leading to degraded 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 onL-isoaspartyl/ (D-aspartyl)-O-methyltransferases (EC 2.1.1.77) These enzymes recognize the isoaspartyl residue and in the presence of S-adenosylmethionine catalyze ester formation
at the isoaspartyl a-carboxyl group in a methyl transfer reaction The ester converts to succinimide, which after hydrolysis is converted toL-aspartate Proteins with isoas-partyl residues can also be degraded by proteolytic enzymes, 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 iso-aspartyl peptidases from Escherichia coli (EcAIII) and Lupinus luteus (LlA) The alignment was generated using CLUSTAL X version 1.81 [59] Identical residues are marked in red, similar in yellow The green arrow indicates the autoproteolytic cleavage site.
Trang 3in 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 gene product could play a role in the metabolism of
glutathione The tripeptide glutathione, c-Glu-Cys-Gly, is
widely used to protect cells against oxidative damage
[52] and in Escherichia coli cells can also be used as an
osmoprotectant [53] Glutathione synthesis depends on the
glutathione synthase genes ghsAB However, transport of
exogenous glutathione into E coli has not been
character-ized According to Parry and Clark [37], expression of the
ybiK gene responded to the presence of cysteine and to
defects in the cysB gene, and the ybiK knockout 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 activity
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 EcAIII
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 activity of these enzymes, and suggest at the
same time a very broad role for potassium-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-biumsp [54] E coli strain DH5a genomic DNA was used
as template in a PCR to obtain the sequence encoding
EcAIII For subcloning and manipulation, the E coli
DH5a strain was used as host in both cases Bacteria were
grown at 37C 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, WI, USA) as described
previously [22] This plasmid was used to transform E coli
BL21-CodonPlus (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¢-GACGAATACCATGGGCAAA
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 (New England Biolabs, 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 The resulting recombinant plasmid 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 (D¼ 0.7 at
600 nm) were induced with 1 mM isopropyl
thio-b-D-galactoside (IPTG) and shaken for 4 h at 300 r.p.m at
37C Cells were centrifuged at 4000 g, 4 C for 15 min and lysed in 100 mL of a solution containing 30 mMTris/HCl,
pH 8.0, 1 mM phenylmethylsulfonyl fluoride, and 15 mM
2-mercaptoethanol The lysate was sonicated and centri-fuged at 4000 g, 4C for 30 min MgCl2solution was added
to the supernatant to a final concentration of 50 mMand the mixture was stirred for 30 min at 4C to remove DNA The solution was centrifuged at 6500 g, 4C for 30 min at 4 C The supernatant was dialyzed against binding buffer containing 5 mM imidazole, 0.5 mM NaCl and 20 mM
Tris/HCl, pH 7.9, and then applied to the HiTrap column prepared as described previously [22] The column was equilibrated with binding buffer and subsequently wash buffer (30 mMimidazole, 0.5 mM NaCl, and 20 mMTris/ HCl, pH 7.9) was applied to remove nonspecifically bound proteins The expected product was eluted with a buffer containing 1Mimidazole, 0.5 mMNaCl, and 20 mMTris/ HCl, pH 7.9 The protein-containing fractions were con-centrated to 4 mL volume and buffer was exchanged to
20 mM Tris/HCl, pH 8.5, 0.1M NaCl, and 10% glycerol using Centricon-YM-30 filters (Millipore, Billerica, MA, USA) The sample was applied 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 molecular mass of 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 (D¼ 0.6
at 600 nm) were induced with 1 mMIPTG and shaken for
3 h at 300 r.p.m at 37C Cells were centrifuged at 4000 g,
4C for 30 min and lysed at 4 C in 100 mL of a solution containing 30 mMTris/HCl, pH 8.0, 10 mMEDTA, 1 mM
dithiothreitol, 0.25 mMphenylmethylsulfonyl 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, 4C for 1 h MgCl2 solution was added to the supernatant to 25 mMconcentration and the mixture was stirred for 1 h at 4C to remove DNA Finally, the solution was centrifuged at 4000 g for 1 h at 4C The protein was purified in a three-step procedure including: fractionation with poly(ethylene glycol) (PEG8K), anion-exchange chromatography, and gel filtration The purifica-tion procedure was carried out at 4C, except for the gel filtration step, which was carried out at 4C for some batches, and at room temperature for others The superna-tant was stirred for 1 h after addition of solid PEG8K to
Trang 49% concentration The suspension was centrifuged at
4000 g, 4C for 30 min 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 mM Tris/HCl, pH 8.5, 150 mM NaCl, and 5%
glycerol, and then dialyzed against the same buffer The
dialyzed protein solution was centrifuged at 8500 g, 4C for
30 min and the supernatant was applied to manually
prepared DEAE-cellulose ion-exchange column or
com-mercially available Mono-Q column (MonoQ HR 10/10,
Amersham Bioscience AB) equilibrated with buffer A After
washing with buffer A, the protein was eluted as a single
peak (at 350 mMNaCl) by application of a 150–500 mM
linear NaCl gradient in buffer A The L-asparaginase
activity was checked using Nessler reagent to detect
ammonia release Protein-containing fractions 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 column (S300 H Sephacryl,
Amersham Bioscience AB, Uppsala, Sweden) equilibrated
with buffer A and the protein was eluted in one peak
corresponding to a molecular mass of 66 kDa Active
fractions were concentrated as described above The purified
enzyme was analyzed by SDS/PAGE and purity was
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 corresponding activity Enzyme activity for
reactions whereL-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 transaminase (GOT) in the presence
of a-ketoglutarate, and (c) formation of NAD+ after
malate dehydrogenase (MDH)-mediated reduction of
oxaloacetate to malate with NADH as cofactor The
decrease of NADH concentration was measured
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 mL of 20 mM
Tris/HCl, pH 7.5, containing 0.1 mM a-ketoglutarate,
0.2 mg of NADH, 12.5 units each of GOT and 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 To determine Kmand kcatvalues, 7–10 different concentrations of a substrate were used, generally ranging from 0.3–10· the Km When aspartate was not the product, we used the Nessler reagent (Aldrich)
to measure the release of ammonia [57] This colorimetric method utilizes alkaline (KOH) solution of an iodide complex of mercury (II) Measurements were performed spectrophotometrically at 414 nm wavelength Calibration curves were prepared using known concentrations of ammonium sulfate Each reaction was performed using
100 lL of the enzyme solution mixed with 900 lL of a solution with a given concentration of a substrate in 20 mM
Tris/HCl, pH 8.5 The mixture was incubated at 37C and 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 H2O (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 of EcAIII was carried out in
25 mMNH4HCO3for 12 h at 37C using 12.5 ng lL)1of trypsin The tryptic peptides were reduced with 10 mM
dithiothreitol for 30 min and alkylated with 55 mM iodo-acetamide for 30 min at room temperature The sample was diluted in 0.1% trifluoroacetic acid and applied to 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 separation was carried out at 0.2 lLÆmin)1 with a linear gradient of acetonitrile from 0–25% (v/v) in 25 min in the presence of 0.05% formic acid The column outlet was coupled to a Q-TOF electrospray mass spectrometer (Micromass) Mole-cular mass analysis was 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 were analyzed using theMASCOTprogram (http://www.matrixscience.com) Additionally, MS meas-urements of intact EcAIII and LlA were performed Protein solution (1 lgÆmL)1) in 0.05% formic acid was injected at 5 lLÆmin)1flow rate to the micro-Z-spray ion source of the mass spectrometer For EcAIII, the MS mass measurements were repeated for protein recovered from crystals that had been stored for over one year The data were analyzed using theMASSLYNXsoftware (Micromass)
Thermostability studies Thermal denaturations were performed on a J-715 spectro-polarimeter (Jasco, Tokyo, Japan) following the ellipticity at
222 nm at 2 nm bandwidth and response time of 4 s in
25 mM Tris/HCl, 300 mM NaCl, pH 7.5, at 1CÆmin)1 heating rate Protein concentration was 100 lgÆmL)1 Analysis of the data was performed by thePEAKFITsoftware (Jandel Scientific Software, San Rafael, CA) Additionally, the effect on enzymatic activity was assessed using the
Trang 5Nessler assay The tested enzyme was preincubated with
L-Asn at different temperatures at 5C intervals, after
which the reaction was stopped and the activity was
measured as described above
Sequence analysis
Amino acid sequences were retrieved from the Swiss-Prot/
TrEMBL databases [58] Multiple sequence alignments for
76 sequences were performed withCLUSTAL XVersion 1.81
[59] The phylogeny was inferred from 423 amino acid sites
Phylogenetic trees were calculated using 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 the Jones–Taylor–
Thornton substitution matrix [61] and amino acid
frequen-cies observed in the sequences under analysis Site-to-site
rate variation was modeled using a gamma distribution with
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 analysis The maximum likelihood trees were
generated by the quartet puzzling procedure with 10 000
puzzling steps and the program settings described above
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 gels It was observed that
on maturation, which was complete in less than 3 days at
4C, the protein underwent an autocatalytic cleavage
characteristic of Ntn-hydrolases [62–64], leading to the
release of two subunits, of 23 kDa (a-subunit) and 14 kDa
(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 homogeneity (Table 1) sufficient for
biochemical and crystallographic studies [36] Even at
intermediate purification steps, complete autoproteolytic
maturation was observed leading to two subunits with
approximate masses of 19 kDa (a-subunit) and 14 kDa
(b-subunit) (Fig 2B) The maturation process was faster at
room temperature and slower at 4C, but could not be
avoided even when the entire purification procedure was
performed very quickly at 4C (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 4C, the a-subunit
was fully converted to the shorter variant Some preliminary
experiments indicate that this secondary cleavage process can be inhibited by Zn2+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 Based on the LlA sequence, the 22893 Da peak can be assigned to the N-terminal subunit 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 mass 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.
Trang 6of 17091 Da and 13852 Da, which can be assigned to the
a- and b-subunits, respectively The positions of these peaks
are exactly the same for the protein recovered from crystals
However, the molecular masses for these two subunits
predicted from the amino acid sequence on the assumption
of autocatalytic cleavage at Gly178–Thr179 (Fig 1) are
18993 and 14419 Da, respectively This suggests that after
the activating event of proteolytic cleavage, the protein
undergoes further proteolysis producing a- and b-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 MS and overall mass measurement allowed us to assign the 13852 Da peak to the b-subunit 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-subunit is not immediately obvious The last residue detected by the MS/MS sequencing is Arg157, but the measured overall mass is higher than for a polypeptide chain terminating with this residue This suggests that there are a few additional amino acids at the C-terminus of the a-subunit, which could not be detected by MS/MS sequencing due to insufficient length of the remaining peptides The closest match is obtained for Ala161 The molecular mass of 17091 Da found in the MS spectrum can
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 studies to be tightly coordinated
by the a-subunit (D Borek & M Jaskolski, unpublished results), also contributes to the total mass
Enzyme assay and determination of kinetic parameters
A summary of the kinetic data is presented in Table 2 The protein concentration of both enzymes was determined using the Bradford method [66] For EcAIII, the calculation
of kcatwas based on molar concentration determined for the shorter versions of both subunits, as obtained from mass spectrometry
Both enzymes could hydrolyze L-asparagine and a b-peptide formed through the Asp side chain Blocking of the a-amino group (as in Gly-L-Asn) or of the a-carboxyl group (as inL-asparagine a-amide) ofL-asparagine resulted
in inactive substrates (Table 2) The latter compound, together withL-glutamine, which is also inactive, demon-strates in addition that the hydrolyzed amide function cannot be either a- or c-, 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)
Fig 3 SDS/PAGE analysis of the secondary cleavage of EcAIII (A)
Incubation at room temperature for 2 h: M, molecular mass marker;
NM, 3 lgÆlL)1protein in 20 m M Tris/HCl, pH 8.5, without any metal
cations; Mg, 10 lL NM + 10 lL 10 m M MgCl 2 ; Ca, 10 lL
NM + 10 lL 10 m M CaCl 2 ; Zn, 10 lL NM + 10 lL 10 m M ZnCl 2 ,
Li, 10 lL NM + 10 lL 10 m M Li 2 SO 4 (B) As above, but with
incubation time of 48 h The protein bands are labeled as follows:
pro-a, intact subunit a (19 kDa); a, shortened subunit a (17 kDa);
b, subunit b (14 kDa).
Trang 7Thermostability studies
Aspartylglucosaminidase from Homo sapiens is
thermo-stable and retains activity up to 80C [67] In contrast,
measurements for the present enzymes indicate lower
thermal stability The melting temperature for EcAIII
obtained by monitoring a CD signal at 222 nm was
59.2C Nessler activity assays are in agreement with this
value and indicate loss of enzymatic activity at 60C for
both proteins
Sequence analysis
The phylogenetic analysis was carried out for 76 amino acid
sequences related to 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 the 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 and bacterial enzymes with isoaspartyl peptidase activity A small fourth branch is established
by eukaryotic enzymes with unknown biochemical chara-cteristics In both the ML and N-J trees the branch 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 splits 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 consists of two groups: bacterial and eukaryotic sequences In contrast
to the archaeal branch, there is congruency with 16S rRNA phylogeny for the eukaryotic sequences The third branch, annotated as isoaspartyl peptidases in Fig 4, is divided
Table 2 Summary of kinetic parameters characterizing the enzymatic activities of EcAIII and LlA with respect to various substrates.
Trang 8into two clusters: one corresponding mostly to plant
enzymes and the other to bacterial enzymes The sequences
in the plant group are closely related
Discussion
In this paper, we have described procedures for successful
expression and purification of two amidohydrolytic 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 LlA reduced the purification procedure
to two steps The His-tag was not removed from the purified LlA protein, as it did not appear to affect the enzyme activity
Fig 4 Bootstrap tree of amino acid sequences of Ntn-amidohydrolases The tree was calculated from the CLUSTAL X version 1.81 alignment [59] Bootstrap values higher than 50% are included Accession numbers of sequences are in parentheses Red branch, mostly archeal asparaginases; blue branch, eukaryotic and bacterial aspartylglucosaminidases; green branch, plant-type asparaginases and their bacterial homologues; brown branch, eukaryotic sequences with unknown biochemical characteristics The sequences of the enzymes studied in this work (LlA and EcAIII) have been underlined.
Trang 9The full sequences of both protein constructs for
expres-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 which 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 to 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
that the native sequence of LlA begins with Met-Gly-,
exactly as in EcAIII (Fig 1)
Both enzymes undergo autoproteolysis, which can be
detected by SDS/PAGE, and form mature heterotetramers
(ab)2, as 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 subunit a of EcAIII (19 kDa) is converted on
incubation to a shorter variant (17 kDa) (Fig 3) This
pattern of maturation of EcAIII, which makes it similar to
human aspartylglucosaminidase [68], was confirmed for
material recovered from dissolved crystals, where 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 MgCl2[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 ZnCl2, which stopped the
truncation of subunit a completely (Fig 3) As the two salts,
MgCl2and ZnCl2, share the same anion, one can identify
the zinc cation as the inhibitor Additionally, this result
indicates that the process of further cleavage is not an
artefact of contamination by metalloproteases, which
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 residue
(Fig 1), a preferred ligand for Zn2+ coordination Zinc
binding by the spacer sequence could change its
conforma-tion and make it unavailable for the second step of
maturation
Tandem mass spectrometry for EcAIII proves that the
first residue of the b subunit is a threonine (Thr179), as
predicted from sequence alignments (Fig 1) with
structur-ally characterized aspartylglucosaminidases This result
allows the identification of Thr179 (and its Thr193 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 kcat/Km 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 andL-Asn [35] Our results suggest that these enzymes serve primarily as isoaspartyl peptidases and that theirL-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 negatively 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 repair reaction In situations where the modification cannot be repaired, the damaged protein should be degra-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 been reported that in E coli the product of the iadA gene is a zinc isoaspartyldipeptidase [41,42] However, this enzyme cannot hydrolyze some of the b-aspartyl dipeptides, and its affinity for b-L-Asp-L-Leu
is relatively low (Km¼ 0.8 mM) Furthermore, E coli mutants deficient in 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, are the missing link of this metabolic pathway The lack of activity for Gly-L-Asn indicates that the enzymes are aminopeptidases In aspartylglucosamini-dases, a free 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 thus appear that discrimin-ation at the a-amino position of the substrates is more connected with the size of the substituent than with a specific pattern of interactions
The fact thatL-Asn amide is not recognized indicates that the specificity of EcAIII and LlA, as well as of aspartylglu-cosaminidases [73], is limited to substrates which possess
a free a-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- and b-amide groups interchanged, does not lead to productive catalysis This, together with the observation of the role of the a-amino substituent [73], might suggest that it can be accommodated in only one way, directing the correct orientation of a substrate in the active site The length of the linker presenting the amide group for hydrolysis is also important, because L-Gln is not hydrolyzed, 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
Trang 10that 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 Obviously, 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 different
Lupinusspecies really serve as asparaginases in developing
seeds [26,27] A possible explanation of the other,
isoas-partyl peptidase, activity is that the seeds have to retain
their ability to grow for a very long time During the
storage period, their proteins can undergo modification
and isoaspartyl peptidase activity is necessary to destroy
the altered proteins and to allow only the healthy seeds to
grow The presence of L-asparaginase activity agrees also
with the usage of L-asparagine, the main storage
com-pound, as a nitrogen source for protein synthesis 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
Cyanobacteriain managing the cyanophycin supply, which
is considered to be a dynamic reservoir of nitrogen for
Cyanobacteria[51]
The true role of EcAIII remains unclear, however Its Km
for L-asparagine is comparable to that of the cytosolic
bacterial-type asparaginase, EcAI (3.5 mM) but is much
higher than the K lAsn
m value for the periplasmic enzyme, EcAII (11.5 lM) [75] Such a low affinity of EcAIII and the
presence of another enzyme with much higher affinity for
L-asparagine argue against the hypothesis that 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 dysfunction
may survive due to the iaaA gene product activity, but the
behavior of an organism with an iaaA knockout has not
been investigated
Recent studies have suggested that enzymes like LlA
and EcAIII might be involved not only in carbon and
nitrogen but also sulfur metabolism [37] Glutathione
(c-Glu-Cys-Gly) catabolism largely concerns the
remobi-lization of cysteine, for example for protein synthesis
during seed storage and during sulfur deprivation The
studies of Parry and Clark suggest that the iaaA gene
product in E coli could be involved in glutathione
transport, as 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 the
c-Glu-Cys dipeptide, as the enzyme 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 part by a subsidy from the Foundation for Polish Science to M J.
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