Báo cáo khoa học: A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for b-alanyl dipeptides Hidenobu Komeda and Yasuhisa Asano docx

A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for b -alanyl dipeptides Hidenobu Komeda and Yasuhisa Asano Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan Many different kinds of microbial hydrolases acting d-stereoselectively on amino acid amides or peptides have been characterized, and some of them have been applied to the production of optically active d-amino acids from the corresponding racemic amino acid amides [1]. The d-stereoselective amidases and peptidases known to date can be classified into four groups based on their primary structures. d-Aminopeptidase from Ochrobactrum anthropi C1-38 [2,3], d-amino-acid ami- dase from O. anthropi SV3 [4,5], alkaline d-peptidases from Bacillus cereus DF4-B [6,7] and AH559 [8], DmpB from O. anthropi LMG7991 [9] and MlrB from Sphingomonas sp. [10] are active site serine hydrolases, which are classified into the penicillin-recognizing Keywords amidase; b-alanine; dipeptidase; DmpA; Pseudomonas sp. Correspondence Y. Asano, Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939–0398, Japan Fax: +81 766 562498 Tel: +81 766 567500 E-mail: asano@pu-toyama.ac.jp (Received 18 March 2005, revised 12 April 2005, accepted 18 April 2005) doi:10.1111/j.1742-4658.2005.04721.x We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene, ramA, from the Pseudomonas sp. MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 (43% identity). The DmpA (called l-aminopeptidase d-Ala-esterase ⁄ amidase) hydrolyzes alanine-p-nitroani- lide, alaninamide, and alanine methylester with a preference for the d-con- figuration of the alanine, whereas the enzyme acts as an l-stereoselective aminopeptidase on a tripeptide Ala-(Gly) 2 , indicating a reverse stereoselec- tivity [Fanuel L, Goffin C, Cheggour A, Devreese B, Van Driessche G, Joris B, Van Beeumen J & Fre ` re J-M (1999) Biochem J 341, 147–155]. A recombinant BapA exhibiting hydrolytic activity toward d-alanine- p-nitroanilide was purified from the cell-free extract of an Escherichia coli transformant overexpressing the bapA gene and characterized. The purified enzyme contained two polypeptides corresponding to residues 1–238 (a-peptide) and 239–366 (b-peptide) of the precursor as observed for DmpA. On gel-filtration chromatography, BapA in the native form appeared to be a tetramer. It had maximal activity at 60 °C and pH 9.0– 10.0, and was inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, Zn 2+ ,Ag + ,Cd 2+ or Hg 2+ . The enzyme hydrolyzed d-alanine-p-nitroanilide more efficiently than l-alanine-p-nitro- anilide the same as DmpA. Furthermore, BapA was found to hydrolyze peptide bonds of b-alanyl dipeptides including b-Ala-l-Ala, b-Ala-Gly, b-Ala-l-His (carnosine), b-Ala-l-Leu, and (b-Ala) 2 with high efficiency compared to d-alanine-p-nitroanilide. b-Alaninamide was also efficiently hydrolyzed, but the enzyme did not act on the peptides containing pro- teinogenic amino acids or their d-counterparts for N-terminal residues. Based on its unique substrate specificity, the enzyme should not be called l-aminopeptidase d-Ala-esterase ⁄ amidase but b-Ala-Xaa dipeptidase. Abbreviations ORF, open reading frame; SD, Shine–Dalgarno. FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS 3075 protein family together with dd-carboxypeptidases involved in peptidoglycan biosynthesis and b-lactamas- es for resistance against b-lactam antibiotics (group 1). Zinc-containing d-alanyl-d-alanine-dipeptidases inclu- ding VanX from vancomycin-resistant enterococci, VanX from the glycopeptide antibiotic producer Strep- tomyces toyocaensis and DdpX from Escherichia coli are considered to be involved in vancomycin-resistance, immunity to the self-produced antibiotic, and cell sur- vival under condition of starvation, respectively [11] (group 2). d-Aminopeptidase, DppA, from Bacillus sub- tilis is a Zn 2+ -dependent self-compartmentalizing pro- tease composed of 10 subunits [12,13]. A thermostable d-alanine amidase homologous to DppA has also been found in Brevibacillus borstelensis BCS-1 [14] (group 3). For the fourth group, DmpA acting on d-alanine- p-nitroanilide (d-Ala-pNA) was found in O. anthropi LMG7991 [9]. Unlike the above d-stereoselective enzy- mes exhibiting strict d-stereoselectivity, DmpA shows a peculiar substrate specificity acting on Ala-p-NA, alaninamide and alanine methylester with a preference for the d-configuration, but acting l-stereoselectively on peptide substrates such as Ala-(Gly) 2 , and has there- fore been called l-aminopeptidase d-Ala-esterase ⁄ ami- dase [15]. The activity of DmpA toward these d- and l-substrates is, however, very weak, suggesting that they are not the true substrates of the enzyme. The bio- logical role of DmpA also remains unclear. We recently found in Pseudomonas sp. MCI3434 a novel amidase, named R-amidase, acting R-stereoselec- tively on piperazine-2-tert-butylcarboxamide and isola- ted the gene coding for the enzyme, ramA [16]. In this study, we determined the nucleotide sequence of the region flanking ramA and found six additional genes. One of them was named bapA and its deduced amino acid sequence showed sequence similarity to the DmpA from O. anthropi LMG7991. The bapA gene was expressed in an E. coli host and the recombinant pro- tein (BapA) acting on d-Ala-pNA was purified and characterized. BapA was found to show a unique sub- strate specificity for b-alanyl dipeptides. Results Characterization of the flanking region of ramA encoding an R-stereoselective amidase from Pseudomonas sp. MCI3434 The R-stereoselective amidase-encoding gene, ramA, had been isolated from the Pseudomonas sp. MCI3434 genome [16]. Sequence analysis of the region down- stream from the termination codon of ramA suggested the presence of an ORF, which was preceded by a Shine–Dalgarno (SD) sequence located within a rea- sonable distance of the presumptive ATG start site. This finding suggested that the ramA gene clustered together with some other genes. The two plasmids pRTB1-Fba and pRTB1-Pst containing fragments cov- ering the flanking region of ramA had been construc- ted previously [16]. Here, we determined the nucleotide sequence of the two inserted fragments to obtain a 6668-bp sequence and found four open reading frames (ORFs) designated ORF1, ORF2 (bapA), ORF3 and ORF4, in the upstream region of the ramA gene and two ORFs designated ORF6 and ORF7 in the down- stream region (Fig. 1). ORF3, -4, and -7 would be transcribed in the opposite direction to the other ORFs. It is interesting to note that the inverted repeat sequence, IR-1 (positions 1351–1497), found in the intergenic region of bapA-ORF3 shows 74% identity over 147-bp with the other sequence, IR-2 (positions 6154–6300), found between ORF6 and ORF7 (Fig. 2). These two inverted repeats may rho-independent transcriptional terminators and not other genomic elements, for example, terminal repeats flanking trans- posable regions, because of absence of direct repeat sequences delineating the two inverted repeats. ORF2 designated bapA was found to be 1098 nucleo- tides long (positions 233–1330) and would encode a protein of 366 amino acids (molecular mass, 38123 Da). A potential ribosome-binding site (AGAA) was located just seven nucleotides upstream from the start codon ATG. In the upstream region of the bapA translational Fig. 1. Schematic view of the inserted fragments of pRTB1-Fba and pRTB1-Pst and structural organization of the 6668-bp DNA containing ORF1, ORF2 (bapA), ORF3, ORF4, ramA, ORF6 and ORF7 from Pseudomonas sp. MCI3434. The location of the ORFs and the direction of transcription (arrows) are indicated. Inverted repeat sequences are indicated by ‘IR-1’ and ‘IR-2’. b-Ala-Xaa dipeptidase from Pseudomonas sp. H. Komeda and Y. Asano 3076 FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS start codon, sequences related to the )35 (TCGTCA) and )10 (TACACT) consensus promoter regions were identified. A blast search indicated that the amino acid sequence deduced from bapA was similar to those of hypothetical protein PA1486 from Pseudomonas aerugi- nosa PAO1 [17], putative d-aminopeptidase PP3844 from Pseudomonas putida KT2440 [18], hypothetical protein PLU2258 similar to d-aminopeptidase from Photorhabdus luminescens (ssp. laumondii) TTO1 [19], aminopeptidase Atu5242 from Agrobacterium tumefac- iens C58 plasmid AT [20] and DmpA (l-aminopeptidase d-Ala-esterase ⁄ amidase) from O. anthropi LMG7991 [15] (Table 1). Figure 3 shows the alignment of the primary structures of BapA from Pseudomonas sp. MCI3434 and its homologous proteins. All the sequences except for DmpA in the figure are hypothet- ical proteins found in the genome sequence but yet to be characterized functionally. DmpA is organized as a homotetramer and each subunit contains two chains (a and b) which result from a probable autocatalytic cleavage of the Gly249-Ser250 peptide bond of the inac- tive precursor polypeptide [15]. Not only this Gly-Ser dyad but also proposed active site residues, Tyr146 and Asn218, the possible two elements of an oxyanion hole [21], were well conserved in the BapA sequence (num- bering of the residues is based on DmpA). Fig. 2. Comparison of the nucleotide sequences of IR-1 and IR-2. Identical bases are marked by asterisks. Table 1. Homology search analysis of seven ORFs in 6668-bp-long DNA region of Pseudomonas sp. MCI3434. P. putida, Pseudomonas put- ida; P. aeruginosa, Pseudomonas aeruginosa; P. luminescens, Photorhabdus luminescens; A. tumefaciens, Agrobacterium tumefciens; O. anthropi, Ochrobactrum anthropi; P. syringae, Pseudomonas syringae; S. meliloti, Sinorhizobium meliloti; E. coli, Escherichia coli; S. flex- neri, Shigella flexneri. ORF Function Amino acid identity (%) Homology Name of ORF Source Accession no. ORF1 Probable periplasmic polyamine-binding protein 54 PP5341 P. putida KT2440 Q88C42 45 PA1410 P. aeruginosa PAO1 A83470 38 PP3845 P. putida KT2440 Q88G80 36 PA2711 P. aeruginosa PAO1 G83306 ORF2 (bapA) b-Ala-Xaa dipeptidase 69 PA1486 P. aeruginosa PAO1 G83460 64 PP3844 P. putida KT2440 Q88G81 45 PLU2258 P. luminescens TTO1 Q7N4R4 44 Atu5242 A. tumefaciens C58 AE3189 43 DmpA O. anthropi LMG7991 Q59632 ORF3 Probable dipeptidase 79 PSTPO1343 P. syringae DC3000 Q887F3 73 PP0203 P. putida KT2440 Q88RC9 ORF4 Probable transcriptional regulator of luxR family 73 PP3847 P. putida KT2440 Q88G78 70 PA3599 P. aeruginosa PAO1 A83196 46 PA2591 P. aeruginosa PAO1 H83320 ORF5 (ramA) R-Stereoselective amidase 73 PP3846 P. putida KT2440 Q88G29 66 PA3598 P. aeruginosa PAO1 H83195 41 PP0382 P. putida KT2440 Q88QV2 37 SMc01962 S. meliloti 1021 Q92MW3 ORF6 Probable periplasmic polyamine-binding protein 74 PP3845 P. putida KT2440 Q88G80 65 PA2592 P. aeruginosa PAO1 A83321 ORF7 Probable nickel ABC transporter 63 PP3346 P. putida KT2440 Q88HL0 51 (NikE) E. coliK-12 C9117E 50 (NikE) S. flexneri 301 Q83J77 H. Komeda and Y. Asano b-Ala-Xaa dipeptidase from Pseudomonas sp. FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS 3077 As summarized in Table 1, the deduced amino acid sequences of the other ORFs, ORF1, ORF3, ORF4, ORF6 and ORF7, showed significant homology with probable periplasmic polyamine-binding protein, prob- able dipeptidase, probable transcriptional regulator of LuxR family, probable periplasmic polyamine-binding protein and nickel ABC transporter, respectively. Comparison of the deduced amino acid sequences of ORF1 and ORF7 with their homologous genes indica- ted that both of the ORFs lack 5¢-terminus, probably coding for about 320 amino acid residues for ORF1 and about 160 amino acid residues for ORF7. The seven proteins encoded in the 6668-bp region of Pseu- domonas sp. MCI3434 showed 38–74% identity with those encoded in P. putida KT2440 genome. Moreover, ORF1-bapA and ORF4-ramA-ORF5 are also in equi- valent cluster arrangement in P. putida KT2440 genome. Production of BapA in E. coli and its purification To express the bapA gene in E. coli, we improved the sequence upstream from the ATG start codon by PCR, with the plasmid pRTB1-Fba as a template as described in Experimental procedures. The resultant plasmid, p2DAPEX, in which the bapA gene was under the control of the lac promoter of the pUC19 vector, was introduced into E. coli JM109 cells. E. coli JM109 harboring pUC19, which was cultured in LB medium supplemented with ampicillin and isopropyl- Fig. 3. Comparison of the amino acid sequences of BapA and homologous pro- teins. Identical and conserved amino acids among the sequences are marked in black and in gray, respectively. Dashed lines indi- cate the gaps introduced for better align- ment. A cleavage site identified in BapA as well as in DmpA is marked by an arrow- head. Proposed active site residues Tyr146 and Asn218 in DmpA are marked by aster- isks. Pse-BapA, BapA from Pseudomonas sp. MCI3434; PA1486, hypothetical protein PA1486 from P. aeruginosa PAO1; PP3844, putative D-aminopeptidase PP3844 from P. putida KT2440; Atu5242, aminopeptidase Atu5242 from Agrobacterium tumefaciens C58 plasmid AT; Oan-DmpA, DmpA ( L-amino- peptidase D-Ala-esterase ⁄ amidase) from O. anthropi LMG7991; PLU2258, hypothet- ical protein PLU2258 similar to D-aminopep- tidase from Photorhabdus luminescens (ssp. laumondii) TTO1. b-Ala-Xaa dipeptidase from Pseudomonas sp. H. Komeda and Y. Asano 3078 FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS b-d-thiogalactopyranoside for 15 h at 37 °C, exhibited no hydrolytic activity toward d-Ala-pNA. When E. coli JM109 harboring p2DAPEX was cultured under the same conditions, the level of hydrolytic activity toward the substrate was 0.776 units per milli- liter of culture, suggesting that the BapA protein was produced in an active form in E. coli. Recombinant BapA was purified from the E. coli JM109 harboring p2DAPEX with a recovery of 10.3% by ammonium sulfate fractionation and DEAE-Toyo- pearl, Butyl-Toyopearl and MonoQ column chromato- graphies (Table 2). The final preparation gave two bands on SDS ⁄ PAGE with molecular masses of  27 and 13 kDa (Fig. 4). These polypeptides were electro- blotted on to a poly(vinylidene difluoride) (PVDF) membrane and submitted to N-terminal amino acid sequencing, which yielded the MRIRE and SIVIT sequences for the 27 and 13 kDa peptides, respectively. These sequences corresponded to residues 1–5 and 239–243 of the deduced amino acid sequence of BapA. This result clearly indicated that the mature enzyme with two polypeptide chains (a and b) was formed by the cleavage of Gly238-Ser239 peptide bond of the 366-residue precursor, as in the case of DmpA from O. anthropi LMG7991. The molecular mass of the native enzyme was about 150 kDa according to gel- filtration chromatography, indicating that the native enzyme was active as a tetramer. The purified enzyme catalyzed the hydrolysis of d-Ala-pNA to d-alanine and p-nitroaniline at 7.73 UÆmg )1 under standard con- ditions. Effects of pH and temperature on stability and activity of BapA The stability of the enzyme was examined at various pH values. The enzyme was incubated at 30 °C for 10 min in the various buffers described in Experimen- tal procedures. Then a sample of the enzyme solution was taken, and the remaining activity of BapA was assayed with d-Ala-pNA as a substrate under standard conditions. The enzyme was most stable in the pH range 6.0–11.0. The stability of the enzyme was also examined at various temperatures. After the enzyme had been preincubated for 10 min, the remaining activ- ity was assayed with d-Ala-pNA as a substrate under standard conditions. It exhibited the following remain- ing activity: 60 °C, 0%; 55 °C, 49%; 50 °C, 87%; 45 °C, 100%; 40 °C, 100%; 35 °C, 100%. The enzyme could be stored on ice without loss of activity for more than one month. The optimal pH for the activity of the enzyme was measured in the buffers used above. The enzyme showed maximal activity at pH 9.0–10.0. The enzyme reaction was also carried out at various temperatures for 1 min in 0.1 m Tris ⁄ HCl (pH 8.0), and enzyme activity was found to be maximal at 60 °C. Above 75 °C, it decreased rapidly, possibly because of insta- bility of the enzyme at the higher temperatures. Effects of inhibitors and metal ions The BapA solution was dialyzed against 20 mm Tris ⁄ HCl (pH 8.0). Various compounds were investi- gated for their effects on the enzyme activity. We meas- ured the activity under standard conditions after incubation at 30 °C for 10 min with various compounds Table 2. Purification of BapA protein from E. coli JM109 harboring p2DAPEX. Total protein (mg) Total activity (U) Specific activity (U ⁄ mg) Yield (%) Cell-free extract 5480 2970 0.54 100 Ammonium sulfate 1310 1810 1.38 60.9 DEAE-Toyopearl 367 2090 5.69 70.4 Butyl-Toyopearl 216 1160 5.37 39.1 MonoQ HR10 ⁄ 10 40 306 7.65 10.3 Fig. 4. SDS ⁄ polyacrylamide slab gel electrophoresis of BapA. Lane 1, molecular-mass standards [phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa)]; lane 2, purified BapA (10 lg). Polypeptides with a molecular mass of 27 kDa and 13 kDa were designated a- and b-peptide, respectively. H. Komeda and Y. Asano b-Ala-Xaa dipeptidase from Pseudomonas sp. FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS 3079 at 1 mm. The enzyme was inhibited by p-chloro- mercuribenzoate (4.9%), N-ethylmaleimide (19.5%), dithiothreitol (37.0%), HgCl 2 (0.8%), ZnSO 4 (1.5%), ZnCl 2 (1.7%), AgNO 3 (5.2%) and CdCl 2 (9.5%): values in parentheses indicate the relative remaining activity. Chelating reagents, e.g. o-phenanthroline, 8-hydroxy- quinoline, ethylenediaminetetraacetic acid, and a,a¢- dipyridyl had no significant effect on the enzyme. Carbonyl reagents such as hydroxylamine, phenylhydr- azine, hydrazine, d,l-penicillamine, and d-cycloserine were not inhibitory toward the enzyme either. A serine protease inhibitor, phenylmethanesulfonyl fluoride, a serine ⁄ cysteine protease inhibitor, leupeptine, and an aspartic protease inhibitor, pepstatin, did not influence the activity. Inorganic compounds such as LiCl, H 2 BO 3 , NaCl, MgSO 4 , MgCl 2 , AlCl 3 , KCl, CaCl 2 , CrCl 3 , MnSO 4 , MnCl 2 , FeSO 4 , FeCl 3 , CoCl 2 , NiCl 2 , CuSO 4 , CuCl 2 , RbCl, Na 2 MoO 4 (NH 4 ) 6 Mo 7 O 24 , SnCl 2 , CsCl, BaCl 2 and PbCl 2 did not affect the activity. Substrate specificity To study the substrate specificity, the purified BapA was used to hydrolyze various amides and peptides and the activity was assayed (Table 3). The enzyme pre- ferred the d-configuration of Ala-pNA, hydrolyzing d-Ala-pNA with 5.8 times higher efficiency than l-Ala- pNA. d-Alaninamide was, however, hydrolyzed by BapA at a much lower rate than d-Ala-pNA, while the hydrolysis of l-alaninamide was below the detection limit. The enzyme did not act on the peptides contain- ing proteinogenic amino acids or their d-counterparts for N-terminal residues. Besides the three substrates which could be hydrolyzed by BapA, dipeptides con- taining b-alanine at the amino terminus, including b-Ala-l-Ala, b-Ala-Gly, b-Ala-l-His (l-carnosine), b-Ala-l-Leu and (b-Ala) 2 were found to be efficiently hydrolyzed by the enzyme. b-Alaninamide was also hydrolyzed by the enzyme. The highest level of activity was observed for b-Ala-l-Ala, being as much as 6.2 times that for d-Ala-pNA. c-Aminobutyryl-l-His (l-homocarnosine) was not hydrolyzed by BapA. These results indicated that the enzyme is specific for N-terminal b-alanyl dipeptides (b-Ala-Xaa). Discussion In this paper, we determined the nucleotide sequence of the flanking region of ramA coding for R-amidase from Pseudomonas sp. MCI3434, and found six ORFs named ORF1, bapA, ORF3, ORF4, ORF6, and ORF7 upstream and downstream of ramA. The amino acid sequence deduced from bapA showed significant similarity to that from dmpA of O. anthropi LMG7991. DmpA has been reported to be a homo- tetrameric enzyme, each subunit of which is composed of two polypeptides generated by a possible autocata- lytic cleavage of a precursor polypeptide. Interestingly, DmpA changes its stereoselectivity depending on the substrate, catalyzing the l-stereoselective hydrolysis of peptides and d-stereoselective hydrolysis of amino acid amides and esters [15]. The gel-filtration chromato- graphy and SDS ⁄ PAGE analysis of the purified BapA revealed that the number of subunits in the native form and the polypeptide composition of each subunit were significantly similar to those for DmpA. The pref- erence for the d-configuration of Ala-pNa and alanin- amide as substrates by BapA was also comparable to that exhibited by DmpA. The substrate specificity of BapA was however, different from that of DmpA with respect to the activity toward peptide substrates. BapA could not hydrolyze l-Ala-(Gly) 2 which is a good sub- strate for DmpA. Furthermore, a broader exploration of the substrate range revealed that BapA acted with much higher efficiency on the b-alanyl dipeptides and b-alaninamide than d-Ala-pNA, and we therefore pro- pose that the enzyme be tentatively called b-Ala-Xaa dipeptidase (EC 3.4.13 ). It would be interesting to investigate whether DmpA can also hydrolyze b-alanyl dipeptides. Table 3. Comparison of the substrate specificity of BapA and DmpA. The following compounds were not substrates for BapA: (Gly) 2 (Gly) 3 , D-Ala-Gly, D-Ala-(Gly) 2 (D-Ala) 2 , D-Ala-L-Ala (D-Ala) 3 (D-Ala) 4 , L-Ala-Gly, L-Ala-(Gly) 2 (L-Ala) 2 , L-Ala-D-Ala, L-Ala-D-Ala-L-Ala, DL-Ala-DL-Asn, DL-Ala-DL-Ile, DL-Ala-DL-Leu, DL-Ala-DL-Met, DL-Ala- DL-Phe, DL-Ala-DL-Ser, DL-Ala-DL-Val, L-Asp-D-Ala, L-Pro-Gly, L-Pro- L-Phe, c-Aminobutyryl-L-His (homocarnosine), Gly-NH 2 , D-Phe-NH 2 , D-Asp-NH 2 , D-Glu-NH 2 , D-Gln-NH 2 , D-Pro-NH 2 , L-Ala-NH 2 , L-Leu-NH 2 , L-Phe-NH 2 , L-Tyr-NH 2 , L-Trp-NH 2 , L-Ser-NH 2 , L-Thr-NH 2 , L-Lys-NH 2 and L-Pro-NH 2 . a Data from reference [15]; N.D. not detectable; N.I. no information. Substrate Activity (UÆmg )1 ) BapA DmpA a D-Ala-pNA 7.7 5.3 L-Ala-pNA 1.3 0.76 D-Ala-NH 2 0.31 0.23 L-Ala-NH 2 N.D. 0.09 D-Ala-(Gly) 2 N.D. 0.04 L-Ala-(Gly) 2 N.D. 1.25 b-Ala- L-Ala 47.4 N.I. b-Ala-Gly 36.0 N.I. b-Ala-NH 2 27.7 N.I. b-Ala- L-His (Carnosine) 27.2 N.I. b-Ala- L-Leu 23.3 N.I. (b-Ala) 2 22.6 N.I. b-Ala-Xaa dipeptidase from Pseudomonas sp. H. Komeda and Y. Asano 3080 FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS Microbial peptidases acting on b-alanyl dipeptides have been studied only in Pseudomonas aeruginosa and Lactobacillus delbrueckii ssp. lactis DSM 7290. Van der Drift and Ketelaars have isolated a bacterium hydro- lyzing b-Ala-l-His (l-carnosine) and identified it as P. aeruginosa [22]. They also investigated some proper- ties of the dipeptidase, called carnosinase, using crude cell-free extracts of the strain. The carnosinase activity in crude extracts was not affected by the addition of EDTA and the enzyme could not hydrolyze c-amino- butyryl-l-His (l-homocarnosine). Comparable observa- tions were made for the purified BapA in this study, suggesting that the carnosinase activity in P. aeruginosa is likely to correspond to the BapA activity in Pseudo- monas sp. MCI3434. On the other hand, pepV has been cloned from the genome of L. delbrueckii ssp. lactis DSM 7290 and identified as a gene coding for carnosin- ase activity [23]. Crude extracts of an E. coli transform- ant producing PepV were subjected to native gel electrophoresis and used for subsequent histochemical staining with various peptides to study the substrate specificity without purification of the enzyme. Although PepV and BapA have some overlap in substrate speci- ficity especially for b-alanyl dipeptides, we also found substrates that could be hydrolyzed by only PepV, such as d-Ala-l-Leu and (l-Ala) 2 . There was no significant homology between the amino acid sequences deduced from pepV of L. delbrueckii and from bapA. BapA is therefore the first example of a highly purified and characterized enzyme specific for b-alanyl dipeptides. Experimental procedures Bacterial strain, plasmids, and culture conditions E. coli JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, D (lac-proAB) ⁄ F¢ [traD36, proAB + , lacI q , lacZDM15]) was used as a host for the recombinant plasmids. Plasmids pRTB1-Fba and pRTB1-Pst [16] containing inserts of 5.3 kb and 2.1 kb, respectively, were used for nucleotide sequen- cing. Plasmid pUC19 (Takara Bio Inc., Shiga, Japan) was used as a cloning vector. Recombinant E. coli JM109 was cultured in LB medium [24] containing 80 lgÆmL )1 of ampi- cillin. To induce expression of the gene under the control of the lac promoter, isopropyl thio-b-d-galactoside was added to a final concentration of 0.5 mm. DNA sequence analysis For routine work with recombinant DNA, established pro- tocols were used [24]. Nested unidirectional deletions were generated from the plasmids, pRTB1-Fba and pRTB1-Pst, with the Kilo-Sequence deletion kit (Takara Bio Inc.). An automatic plasmid isolation system (Kurabo, Osaka, Japan) was used to prepare the double-stranded DNAs for sequen- cing. Nucleotide sequencing was performed using the dide- oxynucleotide chain-termination method [25] with M13 forward and reverse oligonucleotides as primers. Sequen- cing reactions were carried out with a Thermo Sequenase TM cycle sequencing kit and dNTP mixture with 7-deaza-dGTP from Amersham Biosciences K.K. (Tokyo, Japan), and the reaction mixtures were run on a DNA sequencer 4000 L (Li-cor, Lincoln, NE, USA). Both strands of DNA were completely sequenced. The nucleotide sequence data repor- ted in this paper will appear in the DDBJ ⁄ EMBL ⁄ Gen- Bank nucleotide sequence databases with the accession number AB158573. Amino acid sequences were compared with the blast program [26]. Expression of the bapA gene in E. coli A modified bapA gene coding for the DmpA-homologous protein was obtained by PCR. The reaction mixture for the PCR contained in 50 lLof10mm Tris ⁄ HCl, pH 8.85, 25 mm KCl, 2 mm MgSO 4 ,5mm (NH 4 ) 2 SO 4 , each dNTP at a concentration of 0.2 mm, a sense and an antisense primer each at 1 lm, 2.5 U of Pwo DNA polymerase from Roche Diagnostics GmbH (Mannheim, Germany) and 0.1 lg of plasmid pRTB1-Fba as a template DNA. Thirty cycles were performed, each consisting of a denaturing step at 94 °C for 30 s (first cycle 2 min 30 s), an annealing step at 55 °C for 30 s, and an elongation step at 72 °C for 2 min. The sense primer contained an HindIII-recognition site (underlined sequence), a ribosome-binding site (double underlined sequence), and a TAG stop codon (lowercase let- ters) in-frame with the lacZ gene in pUC19, and spanned positions 230–259 in the sequence from GenBank with acces- sion number AB158573. The antisense primer contained an XbaI site (underlined sequence) and corresponded to the sequence from 1320 to 1353. The two primers were as fol- lows: sense primer, 5¢-CACTTG AAGCTTTAAGGAGGA AtagACCATGCGTATCCGTGAGCTTGGCATCACC-3¢; antisen se primer, 5¢-ACGCAA TCTAGAGTCAGCCCTCA GGGGGCTTTCG-3¢. The amplified PCR product was digested with HindIII and XbaI (Takara Bio Inc.), separ- ated by agarose-gel electrophoresis and purified from the gel by use of a QIAquick TM gel e xtraction kit from QIAGEN (Tokyo, Japan). The amplified DNA was inserted downstream of the lac promoter in pUC19, yielding p2DAPEX, and which was then used to transform E. coli JM109 cells. Purification of BapA from E. coli transformant E. coli JM109 harboring p2DAPEX was subcultured at 37 °C for 12 h in a test tube containing 5 mL of LB medium supplemented with ampicillin. The subculture H. Komeda and Y. Asano b-Ala-Xaa dipeptidase from Pseudomonas sp. FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS 3081 (5 mL) was then inoculated into a 2-L Erlenmeyer flask containing 500 mL of LB medium supplemented with ampicillin and isopropyl thio-b-d-galactoside. After a 16-h incubation at 37 °C with rotary shaking, the cells were harvested by centrifugation at 8000 g for 10 min at 4 °C and washed with 0.9% (w ⁄ v) NaCl. All the purification procedures were performed at a temperature lower than 5 °C. The buffer used throughout this purification was Tris ⁄ HCl buffer (pH 8.0) containing 5 mm 2-mercaptoeth- anol and 0.1 mm ethylenediaminetetraacetic acid. Washed cells from the 2.5-L culture were suspended in 100 mm buffer and disrupted by sonication for 10 min (19 kHz; Insonator model 201M; Kubota, Tokyo, Japan). For the removal of intact cells and cell debris, the sonicate was centrifuged at 15 000 g for 20 min at 4 °C. To the cell-free extract was added 5% protamine sulfate, at a concentra- tion of 0.05 g of protamine sulfate to 1 g of protein. After stirring for 30 min, the precipitate formed was removed by centrifugation at 15 000 g for 20 min at 4 °C. The resulting supernatant was fractionated with solid ammo- nium sulfate. The precipitate obtained at 20–40% satura- tion was collected by centrifugation and dissolved in 20 mm buffer. The resulting enzyme solution was dialyzed against 10 L of the same buffer for 24 h. The dialyzed solution was applied to a column (/1.6 · 14 cm) of DEAE-Toyopearl 650M previously equilibrated with 20 mm buffer. After the column had been washed thor- oughly with 20 mm buffer, the enzyme was eluted with 80 mL of 20 mm buffer containing 75 mm NaCl. To the active fractions was added ammonium sulfate to 30% sat- uration. The enzyme solution was applied to a column (/1.6 · 6.5 cm) of butyl-Toyopearl 650M previously equil- ibrated with 20 mm buffer containing ammonium sulfate to 30% saturation. The active fractions were eluted with a linear gradient of ammonium sulfate (30–0% saturation) in 20 mm buffer. The active fractions were combined and dialyzed against 10 L of 20 mm buffer for 12 h. The enzyme solution was applied to a MonoQ HR 10 ⁄ 10 col- umn (Amersham Biosciences K.K) equilibrated previously with 20 mm buffer. After the column had been washed with 30 mL of 20 mm buffer, the enzyme was eluted with a linear gradient of NaCl (0–0.5 m)in20mm buffer using the A ¨ kta-FPLC system (Amersham Biosciences K.K). The active fractions were combined and dialyzed against 10 L of 20 mm buffer for 12 h and used for characterization. N-terminal amino acid sequencing of the purified enzyme was performed at APRO Life Science Institute, Inc. (Tokushima, Japan) with a Procise 494 HT protein sequencing system. Enzyme assay Activity of BapA was assayed routinely at 30 °C by the formation of p-nitroaniline from d-Ala-pNA as follows. A reaction mixture (1.0 mL) containing 5 m md-Ala- pNA, 100 mm Tris ⁄ HCl (pH 8.0), and the enzyme was monitored by the change in absorbance at 405 nm with an Hitachi U-3210 spectrophotometer. One unit of enzyme activity was defined as the amount catalyzing the formation of 1 lmol p-nitroaniline per minute from d-Ala-pNA under the above conditions. Protein was determined by the method of Bradford [27] with BSA as standard, using a kit from Bio-Rad Laboratories Ltd (Tokyo, Japan). To investigate the pH profile of the enzyme stability and activity, the following buffers (final concentration, 100 mm) were used: acetic acid ⁄ sodium acetate (pH 4.0–6.0), Mes ⁄ NaOH (pH 5.5–6.5), potassium phosphate (pH 6.5–8.5), Tris ⁄ HCl (pH 7.5–9.0), ethanolamine ⁄ HCl (pH 9.0–11.0), and glycine ⁄ NaCl ⁄ NaOH (pH 10.0–13.0). The substrate specificity was examined qualitatively by thin-layer chromatography with a solvent system (1-buta- nol ⁄ acetic acid ⁄ water, 4 : 1 : 1, v ⁄ v ⁄ v) first, and then quan- titatively assayed using an HPLC system as follows. The reaction mixture (1 mL) contained 100 lmol Tris ⁄ HCl buf- fer (pH 8.0), 20 lmol substrate and an appropriate amount of the enzyme. The reaction was performed at 30 ° C for 5–30 min and stopped by the addition of 1 mL of ethanol. The amount of product formed in the reaction mixture was determined with an HPLC apparatus equipped with a Sumichiral OA-5000 or OA-5000 L column (/0.46 · 15 cm; Sumika Chemical Analysis Service, Osaka, Japan) at a flow rate of 1.0 mLÆmin )1 , using 2 mm CuSO 4 as a solvent system. The absorbance of the eluate was monitored at 254 nm. Analytical measurements To estimate the molecular mass of the enzyme, the sample (3 lg) was subjected to HPLC on a Superdex 200 HR10 ⁄ 30 column (Amersham Biosciences K.K) at a flow rate of 0.4 mLÆmin )1 with 20 mm potassium phosphate (pH 7.0) containing 150 mm NaCl at room temperature. The absorb- ance of the eluate was monitored at 280 nm. The molecular mass of the enzyme was then calculated based on relative mobility (retention time) using the standard proteins glu- tamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32 kDa), and cytochrome c (12.4 kDa) (products of Oriental Yeast Co., Tokyo, Japan). SDS ⁄ PAGE analysis was performed by the method of Laemmli [28]. Proteins were stained with Brilliant blue R-250 and destained in ethanol ⁄ acetic acid ⁄ water (3 : 1 : 6, v ⁄ v ⁄ v). 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A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for b -alanyl dipeptides Hidenobu Komeda and Yasuhisa Asano Biotechnology Research. substrates for BapA: (Gly) 2 (Gly) 3 , D-Ala-Gly, D-Ala-(Gly) 2 (D-Ala) 2 , D-Ala-L-Ala (D-Ala) 3 (D-Ala) 4 , L-Ala-Gly, L-Ala-(Gly) 2 (L-Ala) 2 , L-Ala-D-Ala,