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: 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: 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 (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 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 (http://www.matrixscience.com). 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. References 1. Borek, D. & Jaskolski, M. (2001) Sequence analysis of enzy mes with asparaginase activity. Acta Biochim . Pol. 48, 893–902. 2. Campbell, H.A., M ashburn , L.T., Boyse, E.A. & Old, L.J. (1967) Two L -asparaginases from Escherichia coli B. Their separation, purification, and antitumor activity. Biochemistry. 6, 721–730. 3. Boyse, E., Old, L., Campbell, H. & Mashburn, L. (1967) Suppression of murine leukemias by L -asparagin ase, incidence of sensitivity a mong leukemias of various types: comparative inhibitory activities of guinea pig serum L -asparaginase and Escherichia coli L -asparaginase. J. Ex p. Me d. 125, 17–31. 4. Gallagher, M.P., Marshall, R.D. & Wilson, R. (1989) Aspar- aginase as a drug for treatment of acute lymphoblastic leukemia. Essays Biochem. 24, 1–40. 5. Mashburn, L. & Wriston, J. (1964) Tumor inhibitory effect of L -asparaginase from Escherichia coli. Arch . Biochem. Biophys. 105, 450–452. 6. Roberts, J., Prager, M.D. & Bachynsky, N. (1966) The antitumor activity of Escherichia coli L -asparaginase. Cancer Res. 26, 2213– 2217. 7. Bonthron, D.T. & Jaskolski, M. (1997) Why a ‘‘benign’’ mut ation kills enzyme activity. Structu re-based analysis of the A 176V mutant of Saccharomyces c erevisiae L -asparaginase I. Acta Biochim. Pol. 44, 491–504. 8. Roberts, J., Holcenberg, J. & Dolowy, W. (1972) Isolation, crys- tallization, andpropertiesofAchromobacteraceae glutaminase- asparaginase with antitumor activity. J. Biol. Chem. 247, 84–90. 9. Ortlund, E., Lacount, M.W., Lewinski, K. & Lebioda, L. (2000) Reactions o f Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibtions and suggests an u nusual catalytic triad Thr- Tyr-Glu. Biochemistry 39, 1199–1204. 10. Sugimoto, H., Odani, S. & Yamashita, S. (1998) Cloning and expression of cDNA encodi ng rat liver 60-kDa lysophospholipase containing an asparaginase-like r egion and ankyrin r epeat. J. B i ol. Chem. 273, 12536–12542. 11. Tumbula, D .L., Becker, H.D., C hang, W.Z. & Soll, D. (2000) Domain-specific recruitment of amide amino acids for protein synthesis. Nature 407, 106–110. 12. Kozak, M. & Jaskolski, M. (2000) Crystallization and preliminary crystallographic studies of a n ew crystal form of Es cherichia coli L -asparaginase (Ser58Ala m utant). Acta Cryst. D. 56, 5 09–511. 13. Lubkowski, J., Wlodawer, A., Ammon, H.L., Copeland, T.D. & Swain, A.L. (1994) S truc tural characterizatio n of Pseu domonas 7A glutaminase-asparaginase. Biochemistry 33, 10257–10265. 14. Lubko wski, J., Wlodawer, A., Housset, D., Weber, I.T., Ammon, H.L., Murphy, K.C. & Swain, A.L. (1994) Refined crystal structure of Acinetobacter glutaminasificans glutam inase-aspar- aginase. Acta Cryst. D. 50, 826–832. 15. Lubkowski, J., Palm, G.J., G illiland, G.L., Derst, C. & Rohm, K.H. (1996) Crystal structure and amino acid sequence of Wolinella succinogenes L -asparaginase. Eur. J. Biochem. 241, 201–207. 16. Lubkowski, J., Dauter, M., Aghaiypour, K., Wlodawer, A. & Dauter, Z. ( 2003) A tomic reso lution s tructure of Er winia chry- santhemi L -asparaginase. Acta Cryst. D. 59, 84–92. 3224 D. Borek et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [...]... (1999) Isolation and characterization of cDNA encoding L-asparaginase from Lupinus luteus (Accession, AF112444) Plant Physiol 119, 1568 23 Gomes, M.A & Sodek, L (1984) Allantoinase and asparaginase activities in maturing fruits of nodulated and non-nodulated soybeans Physiol Plant 62, 105–109 24 Lea, P.J., Farnden, L & Miflin, B.J (1978) The purification and properties of asparaginase from Lupinus species... 2004 Homologous L luteus and E coli amidohydrolases (Eur J Biochem 271) 3225 17 Palm, G.J., Lubkowski, J., Derst, C., Schleper, S., Rohm, K.H & Wlodawer, A (1996) A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant FEBS Lett 390, 211–216 18 Swain, A.L., Jaskolski, M., Housset, D., Rao, J.K.M & Wlodawer, A (1993) Crystal structure of Escherichia. .. Beacham, I.R (1989) Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions Gene 78, 37–46 Supplementary material The following 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 ... covalent modifications of proteins: mechanisms, physiological consequences and clinical applications Matrix Biol 21, 39–52 41 Gary, J.D & Clarke, S (1995) Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli J Biol Chem 270, 4076–4087 42 Jozic, D., Kaiser, J.T., Huber, R., Bode, W & Maskos, K (2003) X-ray structure of isoaspartyl dipeptidase from E coli: a dinuclear zinc... from Escherichia coli Biochim Biophys Acta 1433, 76–86 65 Hirel, P.H., Schmitter, J.M., Dessen, P., Fayat, G & Blanquet, S (1989) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid Proc Natl Acad Sci USA 86, 8247–8251 66 Bradford, M.M (1976) A rapid and sensitive method for the quantitaion of microgram quantities of. .. Isoaspartyl dipeptidase activity of plant-type asparaginases Biochem J 364, 129–136 36 Borek, D & Jaskolski, M (2000) Crystallization and preliminary crystallographic studies of a new L-asparginase encoded by Escherichia coli genome Acta Cryst D 56, 1505–1507 37 Parry, J & Clark, D.P (2002) Identification of a cysB-regulated gene involved in glutathione transport in Escherichia coli FEMS Microbiol Lett... immunological and enzymological survey of asparaginase in seeds of Lupinus Phytochemistry 3, 511–514 26 Lough, T.J., Chang, K.S., Carne, A., Monk, B.C., Reynolds, P.H & Farnden, K.J (1992) L-Asparaginase from developing seeds of Lupinus arboreus Phytochemistry 31, 1519–1527 27 Lough, T.J., Reddington, B.D., Grant, M.R., Hill, D.F., Reynolds, P.H & Farnden, K.J (1992) The isolation and characterisation of a... pp 13–21 Clarendon Press, Oxford 52 Beckerhapak, M & Eisenstark, A (1995) Role of Rpos in the regulation of glutathione oxidoreductase (Gor) in Escherichia coli FEMS Microbiol Lett 134, 39–44 53 Mclaggan, D., Logan, T.M., Lynn, D.G & Epstein, W (1990) Involvement of gamma-glutamyl peptides in osmoadaptation of Escherichia coli J Bacteriol 172, 3631–3636 54 Stratagene (1993) Uni-ZAP XR Library, Instruction... coli L-asparaginase, an enzyme used in cancer therapy Proc Natl Acad Sci USA 90, 1472–1478 19 Sieciechowicz, K.A., Joy, K.W & Ireland, R.J (1988) The metabolism of asparagine in plants Phytochemistry 27, 663–671 20 Lea, P.J & Miflin, B.J (1980) Amino Acids and Derivatives Academic Press, New York 21 Atkins, C.A., Pate, S.J & Sharkey, P.J (1975) Asparagine metabolism – key to the nitrogen nutrition of. .. Rohm, K.H (1992) Probing the role of threonine and serine residues of Escherichia coli asparaginase II by site-specific mutagenesis Protein Eng 5, 785–789 58 Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M.C., Estreicher, A., Gasteiger, E., Martin, M.J., Michoud, K., O’Donovan, C., Phan, I., Pilbout, S & Schneider, M (2003) The SWISSPROT protein knowledgebase and its (Suppl.)TrEMBL in 2003 Nucleic . 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 ,. the expression, purification, and biochemical characterization of two homologous enzymes, with amido- hydrolase activities, of plant (Lupinus luteus potassium- independent asparaginase, LlA) and. 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