Báo cáo khoa học: Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum pptx

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Báo cáo khoa học: Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum pptx

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Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum Tatsuyuki Yamashita 1 , Makoto Ashiuchi 1 , Kouhei Ohnishi 2 , Shin’ichiro Kato 2 , Shinji Nagata 1 and Haruo Misono 1,2 1 Department of Bioresources Science, Kochi University, Nankoku, Kochi, Japan; 2 Research Institute of Molecular Genetics, Kochi University, Nankoku, Kochi, Japan Bifidobacterium bifidum is a useful p robiotic agent exhibiting health-promoting properties a nd contains D -aspartate a s an essential component of the cross-linker m oiety i n t he pepti- doglycan. To help understand D -aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which cata- lyzes the r ac emization of D -and L -aspartate, was purified to homogeneity and characterized. T he enzyme was a mono- mer with a molecular mass of 27 kDa. This is the first report showing th e presence of a m onomeric aspartate racemase. Its e nzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the pres- ence of aspartate, especially the D -enantiomer. The gene encoding the m onomeric as partate r acemase w as cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. T he recombina nt e nzyme w as purified to homogeneity and i ts properties were almost t he same as those of the B. bifidum enz yme. Keywords: aspartate racemase; Bifidobacterium bifidum; D -aspartate; peptidoglycan; probiotic agent. Bifidobacteria, including Bifidobacterium bifidum, have been applied widely as probiotic agents exhibiting health-promo- ting properties. Recent research suggested very interesting functions of bifidobacterial peptidoglycans e.g. redu ction of harmful bacteria and toxic compounds in the intestine, antitumorigenic activities, and immunological enhancement properties [1–3]. B acterial peptidogly cans contain several kinds of D -amino acids [ 4] and a re thought to pro tect cells from protease actions. D -Alanine and D -glutamate occur in the main chains of bifidobacterial peptidoglycans [5]. The cross-linker moieties of B. bifidum contain D -aspartate as the essential component [5]. Two kinds of amino acid racemases, alanine racemase [5] and glutamate racemase [6], have been identified ubiquitously from bacteria, a nd it has been assumed that the former, a pyridoxal 5¢-phosphate (PLP)-dependent amino a cid racemase [5], is i nvolved in D -alanine biosynthesis and the latter, a PLP-independent racemase [6], participates in the supply o f D -glutamate. Most bacterial alanine racemases assemble in a dimer structure [5], w hereas glutamate r acemases are m ainly characterized as monome ric enzymes [6]. On the other hand, aspartate racemase is found in limited organisms [7–11], w hich e ncompass even peptidoglycan-less species, such as archaea a nd mollusks. Recent studies showed two distinct characteristics of aspartate racemases i n t he co- enzyme requirement in catalysis [11,12]. Among them, the PLP-independent aspartate racemase is considered to share structural features and catalytic properties with the gluta- mate racemase [12]. Nevertheless, aspartate racemases are typically dimeric, and neither monomeric nor multimeric aspartate racemase has been ide ntified yet. This paper presents the first identification of PLP-independent monomeric aspartate racemase from B. bifidum NBRC 14252 and its enzymologic characteris- tics, as well as cloning and overexpression of its gene in Escherichia coli. Materials and methods Materials N-tert-butyloxycarbonyl- L -cysteine (Boc- L -Cys) was pur- chased from Novabiochem, La ¨ ufelfingen, Switzerland; o-phthalaldehyde (OPA), from Nacalai Tesque, Kyoto, Japan; a 4-lm Nova-Pack C18 column, from Waters, Milford, MA, USA; a HiPrep Sephacryl S-200 column (1.6 · 60 cm) and a HiTrap Butyl FF column (1.0 mL), from Amersham Bioscience, Uppsala, Sweden; a Bio-Scale Q20 column ( 20 mL) and a protein assay k it, from Bio-Rad, Richmond, CA, USA; a TSK gel G3000SW column, from Correspondence to M. Ashiuchi, Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan. Fax: +81 88 8645200, Tel.: +81 88 8645215, E-mail: ashiuchi@cc.kochi-u.ac.jp Abbreviations:Boc- L -cys, N-tert-butyloxycarbonyl- L -cysteine; BTP, bis-trispropane; IPTG, isopropyl thio-b- D -galactoside; OPA, o-phthalaldehyde; PLP, pyridoxal 5¢-phosphate; Tes, N-tris(hydroxymethyl)-methyl-2-aminoethansulfonic acid. Enzymes: alanine racemase (EC 5.1.1.1); glutamate racemase (EC 5.1.1.3); aspartate racemase (EC 5.1.1.13). (Received 25 August 2004, revised 30 September 2004, accepted 19 October 2004) Eur. J. Biochem. 271, 4798–4803 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04445.x Tosoh, Tokyo, Japan; a PRISM kit, from PerkinElmer, Fremont, CA, USA; restriction enzymes, T4 DNA ligase, and i sopropyl thio-b- D -galactoside (IPTG), from Takara Shuzo, Kyoto, Japan; and a plasmid pATE19, from BioLeaders Corporation, Daejeon, Korea. All other chem- icals were of analytical grade. Bacteria and culture conditions B. bifidum NBRC 14252 was cultured at 37 °Cfor48hin GAM broth (pH 7 .1) comprising 1% peptone, 0.3% soy peptone, 1% protease peptone, 1.35% digested serum, 0.5% yeast extract, 0.22% meat extract, 0.12% liver extract, 0.3% glucose, 0.25% KH 2 PO 4 , 0.3% NaCl, 0.5% soluble starch, 0.03% L -cysteine/HCl, and 0.03% so dium thiogly- colate (Nissui, Tokyo, Japan). Enzyme and protein assays The aspartate racemase activity was estimated by determin- ation of t he antipode formed from either enantiomer of aspartate by HPLC. The reaction mixture (200 lL) com- posed of 0.1 M bis-trispropane (BTP) buffer (pH 7.0), 50 m ML -aspartate, 4 m M dithiothreitol, 1 m M EDTA, and enzyme was incubated at 45 °C for 10 h. The reaction was terminated by a ddition of 50 lLof2 M HCl. After neutralization of the reaction mixture, it was incubated at 25 °C for 2 min with a 0.3 M borate solution (pH 9.0) containing 0.2% Boc- L -Cys and 0.2% OPA. A 2-lL aliquot of the resulting mixture was subjected to a Shimadzu LC-10 HPLC system (Kyoto, Japan) composed of an LL-10AD dual pump, a CBM-10 A control bo x, a n R F-10 A spectrofluorometer, and a DGU-14 A degasser, with a 4-lm Nova-Pack C18 column (3.9 · 300 mm). Other conditions were the same as those described by Hashimoto et al . [13]. One unit of the enzyme was defined as the amount of enzyme that catalyzes the formation of 1 lmol of D -aspartate from L -aspartate p er hour. Protein concentrations were determined using a protein assay kit with bovine serum albumin as a standard. Enzyme purification Harvested c ells of B. bifidum NBRC 14252 (wet weight, 104 g ) were suspended in 200 mL of a standard buffer [10 m M N-tris(hydroxymethyl)-methyl-2-aminoethansulf- onic acid (Tes) buffer (pH 6.5), 4 m M dithiothreitol, and 1m M EDTA] supplemented w ith 0.1 MD -aspartate and 0.1 m M phenylmethanesulfonyl fluoride and then disrupted by sonication on ice for 20 min. The suspension was centrifuged at 12 000 g for 30 min, and the resulting supernatant was dialyzed against the standard buffer (pH 6 .5) and used as the cell extract. All the purification procedures were performed at 4 °C, except heat treatment. The cell extract (595 mL) was subjected to ammonium sulfate fractionation. The 25–50% saturation fraction was dissolved in the standard buffer (pH 6.5) and dialyzed overnight against the same buffer. The enzyme s olution was kept at 60 °C for 30 min in the presence of 0.1 M D -aspartate, and the formed p recipitate was r emoved by centrifugation at 12 000 g for 30 min. The supernatant (179 mL) was subjected to an AKTA prime FPLC system (Amersham Bioscience, Uppsala, Sweden) equipped with a Bio-Scale Q20 column (20 mL) that had been equilibrated with the standard buffer (pH 6.5). After the column was washed with the same buffer and a buffer containing 0.15 M NaCl, the enzyme was eluted with t he buffer containing 0.3 M NaCl. The active fractions were combined, d ialyzed against the standard buffer (pH 6.5) overnight, and con- centrated by ultrafiltration with an Amicon PM-10 mem- brane. The enzyme solution was dialyzed against the standard buffer (pH 6.5) containing ammonium sulfate (15% saturation) and subjected to the FPLC system equipped with a HiTrap Butyl FF column (1.0 mL) that had b een equilibrated with the standard buffer ( pH 6.5) containing ammonium sulfate (15% saturation). After the column had been washed with the same buffer, the enzyme was eluted with a linear g radient of ammonium sulfate (15% to 0% saturation) in the buffer. T he active f ractions were combined, dialyzed against the standard buffer (pH 6 .5) overnight, and concentrated by ultrafiltration with an Amicon PM-10 membrane. NaCl was a dded to the enzyme solution (final concentration, 0.15 M NaCl), and the enzyme solution (2.2 mL) was subjected to the FPLC system equipped with a HiPrep S ephacryl S-200 c olumn (1.6 · 60 cm) that had been equilibrated with the standard buffer (pH 6.5) containing 0.15 M NaCl. The column was developed at t he flow rate of 1.0 mLÆmin )1 with the standard buffer (pH 6.5) containing 0.15 M NaCl. The active fractions were combined, dialyzed against the stand- ard buffer overnight, and concen trated by ultrafiltration with an Amicon PM-10 membrane. Electrophoresis SDS/PAGE was carried out with 12.5% polyacrylamide by the method of Laemmli [14]. Molecular mass determination The molecular mass was determined by HPLC on a TSK gel G300SW column (0.75 · 60 cm) at a flow rate of 0.7 mLÆ min )1 with the standard buffer ( pH 6.5) containing 50 m M D -aspartate a nd 0.15 M NaCl. A calibration curve w as made with the following proteins: glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 k Da), enolase (67.0 kDa), adenylate kinase (32.0 kDa), and cytochrome c (12.4 k Da). The molecular mass of the subunit was estimated by SDS/PAGE. The following marker proteins (Amersham Bioscience, Uppsala, Sweden) were used: rabbit muscle phosphorylase b (97 kDa), bovin e serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 k Da), and a-lactalbumin (14.4 k Da). Isolation of peptides obtained by lysyl endopeptidase- digestion of the enzyme The purified enzyme (1 nmol) was dialyzed against water and lyophilized. The protein was dissolved in 20 lLof8 M urea and incubated at 37 °C for 1 h. To the solution, 60 lL of 0.2 M Tris/HCl buffer (pH 9.0) and 5 pmol of lysyl endopeptidase were added, and the m ixture was incubated at 37 °C for 12 h. The peptides were separated on a Shimadzu HPLC system with a YMC-packed C4 colume (YMC, Ó FEBS 2004 Bifidobacterial aspartate racemase (Eur. J. Biochem. 271) 4799 Kyoto, Japan) using a solvent system of 0.1% trifluoroacetic acid and acetonitrile containing 0.7% trifluoroacetic acid. A 90-min linear gradie nt from 5 to 50% acetonitrile wa s used to elute peptides at a flow rate of 1.0 mLÆmin )1 .The absorbance at 210 nm of the effluent was monitored continuously. The peptides were isolated and lyophilized. Amino acid sequence analysis The N-terminal amino acid sequence of the enzyme and the sequences of the isolated peptides were analyzed with an Applied Biosystems (Forster, CA, USA) model 4 92-protein sequencer linked to a phenylthiohydantoin d erivative ana- lyzer. Genetic manipulation The chromosomal DNA of B. bifidum was prepared by the method of Saito & Miura [15]. Two mixed primers were designed from the N-terminal amino acid sequence of the enzyme and a conserved region between the aspartate racemase genes from Streptococcus thermophilus [16] and Defulfurococcus strain SY [8]: the sense primer [5¢-GG(A, T,G,C)GG(A,T,G,C)ATGGG(A,T,G,C)AC(A,T,G,C)(C, T)T-3¢] and the a ntisense primer [5¢-A(A,G)TA(A,G)TG (A,T,G,C)GC(A,T,G,C)GT(A,G)TT(A,G)CA-3¢]. PCR was performed with ExTaq DNA polymerase (Takara Shuzo, Kyoto, Japan). The nucleotide sequence of the amplified DNA fragment (244 bp) was determined by means of the PRISM kit with an Applied Biosystems 373A DNA sequencer. Next, the inverse PCR of the B. bifidum chromo- somal DNA was carried out as follows. The chromosomal DNA was digested with the restriction enzyme SalI, and the digested fragments were in cubated with T4 DNA ligase to allow self-circulation (or self-ligation). Two single primers were further d esigned from t he determined nucleotide sequence of t he amplified DNA fragment: ASPR1 (5¢-TCCC GATAATCGCCGACGACATCG-3¢)andASPR2(5¢-CG GTTGACCAACCGGATATAGCTT-3¢). PCR was con- ducted using the self-circulated frag ments (as a template DNA) and the two primers, ASPR1 and ASPR2. The nucleotide sequence of the 1-kb region containing a probable structural gene of the enzyme was determined. To establish the overproduction of the enzyme, we first designed two single primers from the nucleotide sequences at both t he immediate u p- and downstream e nds of the structural gene of the enzyme: the sense primer AS-N (5¢-CATG CCATGGGACGACCATTTTTTGCG-3¢), in which an NcoI site (underlined) is incorporated, and the antisence primer AS-C (5¢-CCC AAGCTTCTAGCGGC GGATGGCCTTGGC-3¢), in which a HindIII site (under- lined) i s included. The amplified fragment (726 bp) con- taining the enzyme gene was digested with both restriction enzymes NcoIandHindIII and ligated into the NcoI- HindIII site of pATE19. The constructed plasmid was named pBASPR. The sequence of the enzyme gene in pBASPR was verified in both directions in the same way as described above. Owing to t he re-cloning of the enzyme gene into the Nco I-HindIII site of pATE19, the second amino acid of the enzyme was changed from arginine to glycine. The plasmid pBASPR was introduced into E. coli cells. The E. coli JM109/pPASPR clone constructed was used as the enzyme overproducer. The nucleotide sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under the accession number A B179841. Isolation of recombinant aspartate racemase Cells of the E. coli clone harboring pBASPR, an overpro- ducer o f the enzyme, were inoculated into 1 L of Luria b roth [5] containing ampicillin (50 lgÆmL )1 )andIPTG(2m M )to induce enzyme production. The c ulture was carried out at 37 °C for 16 h. Cells (5 g, wet weight) were suspended in 10 mL of the standard buffer (pH 6.5) supplemented with 0.1 m M phenylmethansulfonyl fluoride and then disrupted by sonication at 4 °C. The cell debris was removed by centrifugation at 12 000 g for 30 mins, and the supernatant was dialyzed against the standard buffer (pH 6 .5) at 4 °C overnight. The enzyme solution ( 19 mL) was used as the cell extract. The recombinant enzyme was isolated with the AKTA prime FPLC system with Bio-Scale Q20 anion- exchange, HiTrap B utyl FF, and HiPrep Sephacryl S-200 gel filtration columns, according to the procedures for the enzyme purification from B. b ifidum NBRC 14252. The purified e nzyme was c oncentrated to  10 mgÆmL )1 and stored at 4 °C. The N-terminal amino acid sequence of the recombinant enzyme was verified with the above protein sequencer. Results Purification of aspartate racemase from B. bifidum The aspartate racemase was purified to homogeneity from cell extracts of B. bifidum NBRC 14252. A summary of the purification is presented in Table 1. The enzyme was purified about 1770-fold from the crude extract with a 2.4% yield. The purified enzyme showed a single b and o n SDS/PAGE (Fig. 1A). Molecular mass and N-terminal amino acid sequence The molecular m ass was estimated t o be 27 kDa by gel filtration on a TSK gel G3000SW c olumn. The molecular mass of the subunit was calculated to be 30 kDa by SDS/ PAGE (Fig. 1A). These r esults indicate t hat the enzyme is a monomer. The 27 kDa fraction from the TSK gel G3000SW column showed the e nzyme a ctivity, and the Table 1. Purification of aspartate racemase from B. bifidum NBRC 14252. Step Total protein (mg) Total activity (units) Specific activity (units/mg) Yield (%) Cell extract 4390 708 0.161 100 Ammonium sulphate (25–45%) 2280 432 0.19 61 Heat treatment 194 243 1.25 34.3 Q20 38.4 193 5.03 27.3 Butyl FF 1.11 39.6 35.7 5.59 Sephacryl S200 0.0593 16.9 285 2.38 4800 T. Yamashita et al.(Eur. J. Biochem. 271) Ó FEBS 2004 gel filtration pattern was not changed with or without 50 m MD -aspartate in the elution buffer. Thus, the active component is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be MRRP- FFAVLGGMGTLATSYI. The amino a cid sequence o f the internal peptide, which was isolated from the lysyl endopeptidase-digest of the enzyme, was ERFHRVGFL- GTMGSRASGVYRQAVEEAGYTFV. pH and thermal stabilities The enzyme was most stable in the pH range of 6.0–7.0 when kept at 30 °Cfor30minin10m M BTP buffers (pH 5.5–9.0) containing 4 m M dithiothreitol and 1 m M EDTA. The enzyme was substantially stable up to 30 °Cin0.1 M Tes buffer (pH 6.5) containing 4 m M dithiothreitol and 1m M EDTA (Fig. 2A). The coexistence of aspartate , however, significantly i ncreased its t hermal stability. The enzyme was stable up to 60 °C in the presence of 40 m M D -aspartate (Fig. 2 A). We further examined the effects of the c oncen trations of L -and D -aspartate on th e e nz yme stability and found that D -aspartate is more effective in the stabilization than L -aspartate (Fig . 2 B). Under the conditions described above, the enzyme showed the maximum activity at pH 7.0–7.5 and 45 °C. Cofactors The enzyme did not require PLP as a coenzyme and w as not inhibited by 10 m M hydroxylamine and 1 m M phenylhydr- azine. The e nzyme was not affected by 10 m M EDTA and 1m M FAD, NAD, NADP, ATP, MgCl 2 ,andMnCl 2 . Thus, the enzyme requires no cofactor. The enzyme was inactivated completely by incubation with 0.1 M p-chloro- mercuribenzoate, 0.1 M HgCl 2 ,1m M N-ethylmaleimide, and 1 m M 5,5¢-dith iobis(2-nitrobenzoate) in 0.1 M BTP buffer (pH 7.0) at 30 °C for 30 min. This fact suggests the essential nature of cysteinyl residue(s) in catalysis. Substrate specificity The enzyme exclusively acts on L -and D -aspartate. Other amino acids, including both enantiomers of glutamate, asparagines, glutamine, alanine, serine, lysine, and arginine, were inactive as substrates. The enzyme was not inhibited by these nonsubstrate amino acids (10 m M ), N-methyl- DL -aspartate (10 m M ), and DL -threo-b-methylaspartic acid (10 m M ). a-Methyl- DL -aspartic acid (10 m M ) slightly inhib- itedtheenzyme(12%). Kinetics The ap parent velocity of the enzymatic aspartate racemiza- tion was measured against various concentrations of both enantiomers of the amino acid. As shown in T able 2, the K m AB Fig. 1. SDS/PAGE of aspartate racemase from B. bifidum (A) and the recombinant enzyme (B). (A) L ane M, the molecular marker proteins; and lane E, the purified aspartate racemase from B. bifidum (1 lgof protein). (B) Lane M, the molecular marker proteins; lane 1, the cell extract of E. coli JM109 (10 lg); lane 2, t he cell extract of the E. coli JM109/pBASPR c lone (10 lg); and lane 3, the enzyme purified from E. coli JM109/pBASPR clone cells (5 lg). Fig. 2. Effects of D -aspartate on the enzyme stability. (A) The enzyme was kept at the indicated tem pera ture for 30 min in a test so lutio n [0.1 M Tes buffer (pH 6.5) containing 4 m M dithiothreitol and 1 m M EDTA] in the absence (d) or th e presence o f 40 m MD -aspartate ( s). (B) The en zyme was incubated at 60 °C for 30 min in the test solution with various concentrations of L - (white bars) or D -aspartate (black bars). The resulting enzyme fractions that w ere in cubated wi th the D -amino acid were s ubjected t o the limit ed filtration w ith Microcon YM -1 0 a nd their enzyme activities we re assayed according to the methods described in Materials and methods. Ó FEBS 2004 Bifidobacterial aspartate racemase (Eur. J. Biochem. 271) 4801 and V max values for L -aspartate were about 14.3 and 13.9 times higher than those for the D -enantiomer. However, the K-value of the reaction was nearly one. Gene cloning and sequencing As described above, the gene encoding the monomeric aspartate racemase w as cloned and sequenced. T he gene encodes a protein c onsisting of 241 amino a cid r esidues (Fig. 3 ). The predicted sequence of the first 20 amino acids was identical with that of the enzyme purified from B. bifidum . The predicted molecular mass (26 784 Da) was in good agreement with that of the enzyme isolated from B. bifidum . Overproduction of the recombinant aspartate racemase and its properties In this study, we s ucceeded in constructing the overproducer of the monomeric aspartate racemase o f B. bifid um,namely, E. coli JM109/pBASPR. As shown in F ig. 1B, the r ecom- binant enzyme was p roduced abundan tly in the E. coli clone and was found primarily as a s oluble e nzyme. A crude extract of the recombinant cells (15.1 unitsÆmg )1 ) had about 94-fold higher enzyme activity than that of B. bifidum NBRC 14252 (0.161 unitsÆmg )1 ). The recombinant enzyme was puri fied to homogeneity with a 20% yield, without ammonium sulfate fractionation and heat treatment as described above. The molecular mass of the recombinant enzyme was e stimated to be 27 kDa in an intact form ( by gel filtration o n a TSK gel G3000SW column) and 3 0 kDa under denatured conditions (by SDS/PAGE analysis) (Fig. 1 B). The spectrophotometric analysis of the recom- binant enzyme r evealed an absorption maximum at 278 nm, and no absorption peak was detected in the region from 300 to 500 nm. The enzymological and kinetic properties of the recombinant enzyme were almost the same as those of the enzyme from B. bifidum NBRC 14252. Discussion The PLP-independent aspartate racemase has only b een characterized from the lactic a cid bacterium, S. thermophi- lus [7,12]. In this study, for the fi rst time, we succeeded in identifying the aspartate racemase from B. bifidum .Enzy- mological properties of aspartate racemase purified from B. bifidum NBRC 14252, such as cofactor independency and susceptibility to thiol-modifying reagents, are similar to those of aspartate racemase from S. thermophilus [7,12]. The B. bifidum enzyme, h owever, is a monomer, w hereas the enzymes from S. thermophilus [7,12] and Pyrococcus hori- koshii [18] are dimers. The predicted amino acid sequence of the enzyme from B. bifidum was similar to tho se of PLP-ind ependent, dimeric aspartate racemases studied so far [8,17,18], as shown in Fig. 3. The similarity scores to the racemases from the lactic acid bacterium S. thermophilus [17], from the sulfur- dependent hyperthermophilic archaeon Desulfurococcus strain SY [8], and from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 [18] were 45, 31, and 32%, respectively. The whole geno me sequence o f Bifidobacterium longum NCC2705 was published recently [16]; t his bacter- ium has a taxonomically close relation with B. bifidum. Table 2. Kinetic parameters of t he m onomeric as partate ra cemase. Activity was measured at 30 °C for 10 h. Parameter Value L -Aspartate K m (m M ) 13.4 V max (lmolÆh )1 Æmg )1 ) 200 V max /K m 14.9 D -Aspartate K m (m M ) 0.94 V max (lmolÆh )1 Æmg )1 ) 14.4 V max /K m 15.3 K eq ( L / D ) 0.974 Fig. 3. Linear alignment of amino acid se- quences of PLP-independent aspartate race- mases. Bb, the monomeric aspartate racemase from B. bifidum; St, the dimeric aspartate racemase from S. thermophilus [17]; D s, the dimeric aspartate racemase from Desulfuro- coccus [8]; and Ph, the dimeric aspartate rac- emase from P. horikoshii [18]. Asterisks indicate identical residues among the four se- quences. Two highly conserved regions that encompass the catalytic cysteinyl residue are indicated by bold underlines. 4802 T. Yamashita et al.(Eur. J. Biochem. 271) Ó FEBS 2004 However, unlike B. bifidum, B. longum does not contain D -aspartate as the essential component of peptidoglycans [4], and no orthologous enzyme protein with the PLP- independent type of aspartate racemase can be found in B. longum NCC2705. In general, enzymatic aspartate racemization is thought to proceed via a Ôtwo-baseÕ mech- anism involving the strictly conserved cysteinyl residue(s) [12,18]. Yamauchi et al. [12] had concluded that the dimerization of aspartate racemase is substantial in catalysis because its composite a ctive site requires a n identical cysteinyl residue from each subunit as the catalytic acid/ base pair. However, Liu et al.[18]recentlyfoundthetwo essential cysteinyl residues in each subunit of the dimeric aspartate racemase and reported that the active sites in the same dimer are independent of each o ther. Our finding of the monomeric a spartate racemase strongly supports the latter hypothesis. Although the bifidobacterial enzyme contains f our cysteinyl r esidues (Cys85, Cys195, Cys197 , and Cys204), its two catalytic residues (Cys85 and Cys204) and the surrounding regions (CN TAH and G CTE) are highly conserved (Fig. 3). Recent research s uggests that the dimerization of aspartate racemase, which a ssembles through t he disulfide bonds, may be involved in an increase in its solubility and thermal stability [18]. On the other hand, we re cently observed another s trategy for elevating t hermal stability during the study of the monomeric, bifidobacterial enzyme (Fig. 2). Kinetic analysis demonstrated that the monomeric enzyme had a comparatively high affinity for D -aspartate (Table 2), suggesting t hat the enzyme bound tightly with the D -amino acid so as to form a confor- mation exhibiting high stability. The detailed analysis of the crystal structure of the dimeric a spartate racemase of P. horikoshii proved that its active sites are arranged in a pseudo-mirror symmetry [18]. This characteristic of the enzyme is probably c oncerned w ith the fact that dimeric aspartate racemases generally reveal little difference in the kinetic parameters for D -and L -aspa- rate [7,8,12]. I n contrast, the d ata in T able 2 point to a significant difference in the kinetic parameters of the monomeric enzyme for t he different e nantiomers of the substrate. Therefore, our observations may contribute to the first identification of nonmirror-symmetric aspartate racemase. Studies on the tertiary structure of the monomeric aspartate racemase are now being conducted in order to understand the general principle in molecular recognition mechanisms of the mirror-symmetric amino acid enantio- mers in amino acid racemases. References 1. Zhang, X.B. & Ohta, Y. (1991) Binding of mutagens by fractions of the cell wall skeleton of lactic acid bacteria on mutagens. J. Dairy Sci. 74, 1477–1481. 2. Se kine, K., Ohta, J., Onishi, M., Tatsuki, T., Shimokawa, Y., Toida, T., Kawashima, T. & Hashimoto, Y. (1995) Analysis of antitumor p roperties of effector cells stimulated with a cell wall preparation (WPG) of Bifidobacterium infantis. Biol. Pharm. Bull. 18, 148–153. 3. Sasaki, T., Samegai, T. & Na mioka, S. (1996) Phagocytosis of splenetic neutrophils of mice enhanced by orally administered peptidoglycan from Bifidobacterium thermophilum. J. Vet. Med. Sci. 58, 85–86. 4. Schleifer, K.H. & K andler, O. (1972) Peptidogl ycan types of bacterial cell walls and their taxonomic implic ations. Bacteriol. Rev. 36, 407–477. 5. Yamashita,T.,Ashiuchi,M.,Ohnishi,K.,Kato,S.,Nagata,S.& Misono, H. (2003) Molecular characterization of alanine racemase from Bifidobacterium bifidum. J. Mol. Catal. B: Enzym. 23, 213– 222. 6. Ashiuchi, M., Soda, K. & Misono, H. (1999) Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme. Bios ci. Biotechnol. Biochem. 63, 792–798. 7. Okada,H.,Yohda,M.,Giga-Hama,Y.,Ueno,Y.,Ohdo,S.& Kumagai, H. (1991) Distribution and purification of aspartate racemase in lactic acid bacteria. Biochim. Biophys. Acta 1078, 377–382. 8. Yohda,M.,Endo,I.,Abe,Y.,Ohta,T.,Iida,T.,Maruyama,T.& Kagawa, Y. (1996) Gene for aspartate racemase from the sulfur- dependent hyperthermophilic archacum, Desulfurococcus strain SY. J. Biol. Chem. 271, 22017–22021. 9.Matsumoto,M.,Honmma,H.,Long,Z.,Imai,K.,Ida,T., Maruyama,T.,Aikawa,Y.,Endo,I.&Yohda,M.(1999) Occurrence of free D -amino acids and aspartate racemases in hyperthermophilic archaea. J. Bacteriol. 181, 6560–6563. 10. Long, Z., Lee, J A., Okamoto, T., Sekine, M., Nimura, N., Imai, K., Yohda, M., Maruyama, T., Sumi, M., Kamo, N., Yamagishi, A., Oshima, T. & Homma, H. 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( 1970) C leavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 15. Saito, M. & Miura, K. (1963) P reparation o f tran sforming d eoxy- ribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619–629. 16. Sc hell, M.A., Karmirantzou, M., Snel, B., Vilanova, D., Berger, B., Pessi, G., Zwahlen, M.C., Desiere, F., Bork, P ., Delley, M., Pridmore, D. & Arigon i, F. (2002) The g en ome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Pr oc. Natl Acad. Sci . USA 99, 1 4422– 14427. 17. Yohda, M., Okada, H. & Kumagai, H. (1991) Molecular cloning and n ucleot ide sequencing of the aspartate racemase gene f rom lactic acid bacteria Streptococcus thermophilus. Biochim. Biophys. Acta 1089, 234–240. 18. Liu,L.,Iwata,K.,Kita,A.,Kawarabayasi,Y.,Yohda,M.& Miki, K. (2002) Crystal structure of aspartate racemase from Pyrococcus h o rikoshii OT3 and its implications for molecular mechanism o f P LP-indep ende nt ra cemiza tion. J. Mol. Biol. 319, 479–489. Ó FEBS 2004 Bifidobacterial aspartate racemase (Eur. J. Biochem. 271) 4803 . protein sequencer. Results Purification of aspartate racemase from B. bifidum The aspartate racemase was purified to homogeneity from cell extracts of B. bifidum NBRC 14252. A summary of the purification. Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum Tatsuyuki Yamashita 1 , Makoto

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