Báo cáo Y học: Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N -acyl-D-amino acid amidohydrolase responsible for D-amino acid production pdf

Eur J Biochem 269, 4868–4878 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03190.x Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N -acyl-D-amino acid amidohydrolase responsible for D-amino acid production Pei-Hsun Lin1, Shiun-Cheng Su1, Ying-Chieh Tsai2 and Chia-Yin Lee1 Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan; 2Graduate Institute of Biochemistry, Yang-Ming University, Taipei, Taiwan An N-acyl-D-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-D-methionine The bacterium was classified as Variovorax paradoxus by phylogenetic analysis The gene was cloned and sequenced The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids The V paradoxus N-D-AAase showed significant amino acid similarity to the N-acyl-D-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44–56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity) After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography A homogenous protein with a specific activity of 516 mg)1 D-Amino acids are important materials for chiral chemical synthesis of such things as semi-synthetic antibiotics [1–3], bioactive peptide [4–6], pyrethrods, pesticides and some food additives such as altimate [7,8] They can also be used to synthesize D-configuration specific D-amino acid derivatives [9,10] D-amino acids also are important constituents of eubacterial cell walls [11] They are found in microorganisms, plants and animals and their function and physiological roles have been investigated and identified [12,13] N-acyl-D-amidohydrolase (EC.3.5.1.81, N-D-AAase) is an enzyme capable of catalysing the hydrolysis of N-acylD-amino acids to yield the corresponding D-amino acid and the organic acid They have been found in a number of bacterial species, including members of the Alcaligenes, Strep- Correspondence to C.-Y Lee, Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec 4, Roosevelt Road., Taipei 106, Taiwan Fax: +886 2366 0581, Tel.: +886 2363 0231, extn 2816 E-mail: m477@ccms.ntu.edu.tw Abbreviations: N-D-AAase, N-acyl-D-amidohydrolase; HSL, homoserine lactone; C4-HSL, N-butanoyl-homoserine lactone Enzyme: N-acyl-D-amidohydrolase (N-D-AAase, EC.3.5.1.81) (Received 30 June 2002, revised 14 August 2002, accepted 20 August 2002) was finally obtained After peptide sequencing by LC/MS/ MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 °C, respectively After 30 heat treatment at 45 °C, between pH and pH 8, 80% activity remained The N-D-AAase had higher hydrolysing activity against N-acetyl-D-amino acid derivates containing D-methionine, D-leucine and D-alanine and against N-chloroacetyl-D-phenylalanine Importantly, the enzyme does not act on the N-acetyl-L-amino acid derivatives The enzyme was inhibited by chelating agents and certain metal ions, but was activated by mM of Co2+ and Mg2+ Thus, the N-D-AAase from V paradoxus can be considered a chiral specific and metaldependent enzyme Keywords: N-acyl-D-amino acid amidohydrolase; D-amino acid; LC/MS/MS; Variovorax paradoxus tomyces, Pseudomonas, Stenotrophomonas, Amycolatopsis and Sebekia [14–22] So far, all N-D-AAases characterized consist of monomeric proteins of  45–55 kDa except for the Pseudomonas sp 1158 enzyme which has a molecular mass of 100 kDa and the Amycolatopsis enzyme which has a molecular mass of 36 kDa They have similar optimal temperatures (45–50 °C) and pHs [7,8] but show a variety of different specific activities towards different substrates The enzyme is inhibited by metallic ions such as Zn2+, Hg2+, Cu2+ and by EDTA Notably, the enzymes purified from Streptomyces olivaceus and Amycolatopsis orientalis IFO12806 are activated by Co2+ (1 mM) Some purified enzymes have been found to contain between 2.06 g and 2.61 g Zn per mole and it is considered that zinc ions may play a role in the catalytic activity and stability of the enzyme structure [23–25] Up to this point, it is not clear what the function of N-D-AAase is in bacteria, and gene sequence information is available only from Alicaligenes species [26–28] D-amino acids are very important for the synthesis of intermediate chiral compounds as mentioned earlier and some reports have described enzymatic methods for the synthesis of D-amino acids [29–32], including the coupling in a process of N-D-AAase and N-acylamino acid racemase However, some N-D-AAases isolated from bacteria have some L-aminoacylase activity [18,33] Therefore, it is necessary to avoid L-aminoacylase interference if the enzyme is to be Ó FEBS 2002 used in industrial applications In this study, a strain, Iso1, with N-D-AAase enzyme activity, was isolated from the environment and the gene for the enzyme was cloned and then sequenced The recombinant protein, N-D-AAase, was also produced in the Escherichia coli, purified and characterized MATERIALS AND METHODS Bacterial strains, plasmids and conditions Variovorax paradoxus Iso1 was isolated from an environmental situation containing N-acetyl-D-methionine It was grown at 30 °C in TSB (Difco) medium and used as a source of its chromosomal DNA E coli XL1-Blue [34] and E coli Top10 grown at 35 °C in Luria–Bertani broth (Difco) were used as the host for gene cloning and expression Luria–Bertani medium supplemented with 100 mgỈmL)1 ampicillin (Sigma) was used for plasmid maintenance Two plasmids pBluescript II KS(+) (Stratagene) and pTrcHis2A (Invitrogen) were used as gene cloning and expression vectors, respectively For protein expression, E coli Top10 containing recombinant plasmids was grown in 2YT medium supplemented with 200 mgỈmL)1 ampicillin Under the trc promoter and lacq repressor of pTrcHis2A, isopropyl thio-b-D-galactoside was added to a final concentration of mM Materials, enzymes and chemicals Restriction enzymes and T4 DNA ligase were from New BioLabs and Gibco BRL Pfu DNA polymerase and alkaline phosphatase were from Promega and Boehringer Mannheim, respectively D-Amino acid oxidase (EC 1.4.3.3) from porcine kidney and horseradish peroxidase were purchased from Sigma Chemical Co DEAE-Toyopearl 650 M and Butyl-Toyopearl 650 M were from Tosoh (Tokyo, Japan) FPLC-Mono Q was from Pharmacia Substrates and standards were from commercial sources such as Sigma or Bachem All other reagents were the highest grade available 16S rDNA gene sequence analysis The nucleotide sequence of the 16S rDNA from strain Iso1 was amplified by PCR using proof reading Pfu DNA polymerase The universal primers 5F (5¢-TGAAGAGTTT GATCATGGCT-3¢) and 1540R (5¢-AAGGAGGTGAT CCAACCGCA-3¢) numbered according to the E coli 16S rRNA sequence were used The PCR product was purified and ligated into the p-GEM-T Easy Vector system (Promega) [35] DNA sequencing was carried out using an ABI Prism 3770 DNA sequencer (Perkin Elmer) Comparison with other 16S rDNA sequences was performed by the BLAST program [36] against GenBank The sequence alignment analysis was carried out using CLUSTAL W [37] The PHYLIP software package was used for phylogenetic analysis and TREE VIEW32 was used to view the phylogenic trees [38] The reliability of the each tree node was confirmed by bootstrapping (1000 trees) and a consensus tree was constructed using SEQBOOT and CONSENSE from the PHYLIP package The GenBank accession number for the strain Iso1 is AY127900 V paradoxus N-D-AAase gene (Eur J Biochem 269) 4869 Cloning of V paradoxus N -D-AAase gene Recombinant DNA technology was carried out by the standard methods of Sambrook et al [39] Total genomic DNA was prepared from V paradoxus Iso1 by a modified method, and partially digested with Sau3AI The kb to kb DNA fragments were purified from 1.2% (w/v) lowmelting-point agarose gels (FMC SeaPlaque agarose), and eluted by heating to 67 °C followed by phenol extraction twice and ethanol precipitation The DNA was ligated into BamHI-digested and dephosphorated pBluescript II KS(+) using T4 DNA ligase Competent cells E coli XL1-Blue were transformed by electroporation according to the protocol manual of the Gene Pulser II (Bio-Rad) White colonies were selected into an ELISA microplate containing 50 lL Luria–Bertani medium supplemented with 100 lgỈmL)1 ampicillin in each well using a sterile toothpick and incubated at 37 °C overnight The transformants were screened for enzyme activity by adding to each well 10 lL mgỈmL)1 lysozyme and incubating at 37 °C for 30 This was followed by the addition 110 lL 25 mM N-acetyl-D-methionine and incubation at 40 °C overnight Then, 40 lL of the colour reagent (50 mM Tris/HCl pH 7.5, mL)1 D-amino acid oxidase, 10 mL)1 horseradish peroxidase, lL phenol, 0.2 mgỈmL)1 4-aminoantipyrine) was added and the plate was incubated at room temperature for 10–20 Wells positive for the enzyme should develop a red colour and V paradoxus Iso1 was used as the positive control, whereas E coli containing pBluescript II KS(+) plasmid was used as the negative control Any positive clones were then confirmed by replacing the substrate with buffer The one positive E coli transformant contained a 12.3-kb plasmid, designated pBKdamD4 Southern analysis Chromosomal DNA completely digested by EcoRI or HindIII was separated on a 0.8% agarose gel DNA fragments were transferred onto Zeta-Probe membrane [39] A SacI–PstI DNA fragment of pBK-damH1 was labelled using a random-primer labelling kit (Roche) with [a-32P]dCTP After hybridization at 65 °C and washing, the membrane was exposed to X-ray film at )70 °C Nucleotide and amino acid sequence analysis For sequencing, the N-D-AAase gene, pBK-damD4 was digested with various restriction enzymes and subcloned into pBluscript II KS(+) to obtain the clone pBK-damH1 that carried the smallest insert fragment that retained high enzyme activity The pBK-damD4 and pBK-damH1 were used as sequencing templates to double confirm both strands of the gene The nucleotide sequencing was carried out using an ABI Prism 3770 DNA sequencer (Perkin Elmer) The nucleotide sequence was analysed by using the DNASIS (Hitachi, Japan) and GNEYTEX (Hitachi, Japan) programs The amino acid sequence was compared with known protein sequences in the nucleotide/protein sequence databases by the BLAST program from the Swiss-Prot database Sequence alignment was carried out using the program CLUSTAL W [37] The accession number of the gene reported in this paper is AY126714 Ó FEBS 2002 4870 P.-H Lin et al (Eur J Biochem 269) Construction of a plasmid to produce the recombinant protein N -D-AAase in the E coli A DNA fragment coding for the N-D-AAase was obtained by PCR using the Pfu DNA polymerase (Promega) PCR amplification was carried out as follows: 94 °C for followed by 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 30 s at 72 °C and then a further extension at 72 °C The PCR mix before amplification contained a final concentration of 5% acetylamine The PCR product was purified, digested with EcoRI and HindIII, ligated into pTrcHis2A and finally transformed into E coli Top10 The plasmid pTrcHis2A carrying the whole of the N-D-AAase coding sequence was digested with EcoRI and NcoI, treated with mung bean nuclease and self-ligated This step was to optimize the distance between the vector-borne ShineDalgarno sequence and the N-D-AAase start codon The sequence upstream of the N-D-AAase gene is: 5¢…AGGACAGACGAATG…3¢ (The Shine-Dalgarno sequence and start codon are in bold) The recombinant plasmid was named pTrc2A-damA3 without His-tag Expression and purification of the N -D-AAase from the E coli transformant The E coli Top10 harbouring the pTrc2A-damA3 was subcultured at 35 °C for 8–12 h in a test tube containing mL 2YT medium supplemented with 200 lgỈmL)1 ampicillin The subculture was diluted : 50 into a 500-mL flask containing 150 mL of the same medium and incubated at 35 °C, 150 r.p.m At OD600 ¼ 0.6, isopropyl thio-bD-galactoside was added to a final concentration of mM and the culture was quickly shifted to a temperature of 20 °C and induced for 30 h with shaking The cells from a total of L culture were harvested by centrifugation (8000 r.p.m., 10–20 min) and washed twice with 50 mM Tris/HCl pH 7.5 All purification procedures were performed at °C except the FPLC-Mono Q chromatography, which was carried out at room temperature The pellets were resuspended in lysis buffer (50 mM Tris/HCl, 10% glycerol, 0.01% 2-mercaptoethanol, mM phenylmethanesulfonyl fluoride pH 7.5) and disrupted by a French press cell (12 000–20 000 psi), followed by the immediate addition of mM phenylmethanesulfonyl fluoride and protease inhibitor (Merck, 10 gỈmL)1 E coli) Cell debris were removed by centrifugation (12000 r.p.m., 1–2 h), heated to 40 °C for 15 min, and then centrifuged to remove any unstable protein After dialysis with buffer A (50 mM Tris/HCl, 10% glycerol and 0.01% 2-mercaptoethanol, pH 7.5), the crude protein was loaded onto a DEAE-Toyopearl 650 M column (2.6 · 15 cm) pre-equilibrated with buffer A After washing with 2.5 bed vols buffer A, the adsorbed protein was eluted stepwise with buffer A over a linear gradient containing 0–0.25 M NaCl The pooled active fractions were brought to 20% ammonium sulfate saturation and applied to a Butyl-Toyopearl 650 M column (1.6 · 7.5 cm) pre-equilibrated with buffer B (buffer A containing 20% ammonium sulfate) After washing with bed vols buffer B, the enzyme was eluted with the buffer A containing 15% saturated ammonium sulfate The active fractions were combined, concentrated by Centriprep YM-10 (Amicon) and applied to a column of Sephacryl HR S-200 equilibrated with buffer C (50 mM Tris/HCl, 0.15 M NaCl, 0.01% 2-mercaptoethanol, pH 7.5) The eluted fractions were made up to a final concentration of 10% glycerol and the active fractions were combined for dialysis against buffer C containing 10% glycerol then concentrated using a Centriprep YM-10 Finally, the sample was added to a FPLC Mono Q (Pharmacia) at a flow rate of 0.5 mLỈmin)1 All fractions were assayed for enzyme activity and the active fractions were further analysed by Western blotting Enzyme activity assay The standard reaction mixture (0.5 mL) for the determination of N-D-AAase activity contained 50 mM Tris/HCl pH 7.5 and 25 mM N-acetyl-D-methionine to which an appropriate amount of the enzyme was added The reactions were incubated at 40 °C for 10–30 and then stopped by heat treatment at 100 °C for 10 D-methionine was determined using the colorimetric assay carried out as follows: 100 lL of the enzyme assay solution was mixed with 60 lL of 50 mM Tris/HCl pH 7.5, 20 lL )1 D-amino acid oxidase (3 mL ) and 20 lL colorimetric solution containing peroxidase (10 mL)1), 4-aminoantipyrine (0.04 lgỈmL)1) and 0.8 lL phenol This was then incubated at room temperature for 10 and measured at 520 nm using D-methionine as the standard Protein concentration was determined by the Bradford method with BSA as the standard [40] One unit of N-D-AAase enzyme activity was defined as the formation of lmol )1 D-methioninmin SDS/PAGE and Western analysis The proteins were separated by SDS/PAGE (10% acrylamide) as described by Laemmli [41] For Western blotting, the proteins were transferred to poly(vinylidene fluoride) membrane using 10 mM Caps containing 10% methanol by a semidry transfer device (Pharmacia) for 1–2 h at 50 mA and V After transfer, the membrane was immersed in M urea-PBST (phosphate buffer/saline/Tween-20) solutions with overnight shaking The membrane was washed three times with PBST for 10 then blocked with GelatinNET (NaCl/EDTA/Tween-20) for 1–2 h The primary antibody (1 : 20000 anti N-D-AAase from Alicaligenes faecalis DA1) was incubated with the membrane at room temperature for h, and then washed three times The diluted second antibody (1 : 5000 anti-rabbit horseradish peroxidase) was then added and the membrane was incubated for h followed by three washes Following the protocol supplied with the peroxidase substrate kit (Vector Lab, Inc.), signal bands appeared after the membrane was incubated at room temperature for 5–20 Peptide sequencing by LC/MS/MS analysis and isoelectric focusing After separation by SDS/PAGE, the proteins were detected by staining the gel with Coomassie blue R250 and then destained Proteins to be identified were excised from the gel and processed for mass spectrometric analysis by the ion trap mass spectrometry processes including in-gel reduction, S-carboxyamidomethylation, and trypsin digestion The reaction mixture was then introduced directly into the Ó FEBS 2002 V paradoxus N-D-AAase gene (Eur J Biochem 269) 4871 electrospray ionization (ESI) source of a quadrupole ion trap mass spectrometer (Finnigan LCQ) by a reverse phase microcapillary column [42] Peptides were eluted at a flow rate of 500 nLỈmin)1 and the MS/MS spectra of each peptide was identified by comparison with known peptide sequences [43] IEF determination was performed using a Pharmacia Ampholine PAGplate (pH 3–9 gradient gel) using a broad pI calibration kit Influences of temperature and pH on enzyme activity For the determination of the optimal temperature of the enzyme, the reaction was carried out at 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 °C and the enzyme activity measured as described above Pre-incubation at the indicated temperature for 30 was followed by the determination of the residual enzyme activity was used as a measure the thermostability of the enzyme The substrate 25 mM N-acetyl-D-methionine in various buffers was used to determine the optimal pH The buffers used were: 50 mM acetate buffer (pH 4.0–5.6), phosphate buffer (pH 6.0–7.2), Tris/HCl buffer (pH 7.0–8.6) and glycine/NaOH buffer (pH 8.8–10.2) To measure enzyme stability at the various pH values, the enzyme was preincubated at 35 °C for 30 in the different buffers and the residual enzyme activity was measured by the colorimetric assay Influences of chelating reagents and metal ion on enzyme activity Chelating reagents and metal ions were added to the enzyme reaction which was then preincubated at 35 °C for 30 followed by the addition of 25 mM N-acetyl-D-methionine and the residual enzyme activity was measured by the Chirobiotic T HPLC method using D-methionine as standard [44] The test concentration of chelating reagents and metal ions used for assay were mM and 10 mM, respectively Substrate specificity analysis Various substrates (25 mM) were added to the enzyme in the standard reaction described previously and incubated at 40 °C for 20 The amount of D-amino acids produced was determined by the Chirobiotic T HPLC method and the appropriate D-amino acids were used as the standards RESULTS Identification and phylogenetic analysis of the strain Iso1 The nucleotide sequence of the 16S rRNA of strain Iso1 was determined and compared with other bacterial 16S rRNA sequences corresponding to the E coli 16S rRNA from positions 28–1489 A BLAST search of GenBank showed that the strain Iso1 had the highest similarity to various V paradoxus species The 16S rRNA of strain Iso1 was 99% similar to those of other V paradoxus strains By using the neighbour-joining, maximum-parsimony and maximumlikehood (Fig 1) methods from PHYLIP and testing the resulting trees using bootstrap analysis, the strain Iso1 specifically associated with V paradoxus strains 100% of the time (1000 bootstraps) Other biochemical activity Fig Phylogenetic relationships of the 16S rDNA sequence of the strain Iso1 with other bacteria The GenBank accession numbers for the organisms used in this analysis were as follows: V paradoxus MBIC3839, AB008000; V paradoxus IAM12373, D88006; V paradoxus E4C, AF209469; V paradoxus VAI-C, AF250030; Aquaspirillum delicatum, AF078756; Xylophilus ampelinus, AF078758; Acidovorax facilis, AF078765; Rhodoferax fermentans, RHYFR2D; Hydrogenophaga taeniospiralis, AF078768; Aquaspirillum sinuosum, AF078754; Comamonas acidovorans, AF149849; Ralstonia campinensis, AF312020; Leptothrix mobilis, X97071; Brachymonas denitrificans, D14320; Pandoraea pnomenusa, AF139174; Burkholderia brasilensis, AJ238360; E coli, A14565 The phylogenetic tree was based on the alignment of the 16S rDNA sequences The 16S rDNA sequence of E coli was used as an outgroup analyses [45] and Biolog system kit (Biolog Inc.) identification also showed that the strain Iso1 was V paradoxus For example, the stain Isol was positive for catalase, oxidase and nitrate reduction but negative of hydrolysis for gelatin and starch Therefore, according to all above analysis results, the strain Iso1 was clearly a strain of V paradoxus Cloning and nucleotide sequencing analysis of the N -D-AAase from V paradoxus Iso1 A V paradoxus Iso1 total genomic library was constructed in E coli XL1-Blue One positive clone (pBK-damD4) was found among 1840 clones tested and it developed a faint red 4872 P.-H Lin et al (Eur J Biochem 269) Ó FEBS 2002 colour in the ELISA microplate N-D-AAase enzyme activity assay system after blue-white selection The pBKdamD4 plasmid contained an insert of  kb and this was used for Southern hybridization and subcloning to generate deletion plasmids for nucleotide sequencing The Southern hybridization analysis indicated that the insert fragment was derived from V paradoxus chromosomal DNA (data not shown) At the same time, degenerate primers for the N-DAAase gene were developed using alignment analysis of the other N-D-AAase protein gene sequences in the GenBank database A single band was obtained after PCR amplification with these degenerate primers using the plasmid pBK-damD4 as DNA template (data not shown) and this was used to completely re-sequence the N-D-AAase gene The nucleotide sequence of the open reading frame of the N-D-AAase gene was 1467 bp and encoded 488 amino acid residues with a predicted molecular weight of 52 798 (DNASIS software) (Fig 2) The GC content was about 64.21%, which is consistent with the genome of V paradoxus (66.8–69.4%) A poorly conserved Shine-Dalgarno sequence and three possible )10 and )35 regions were predicted in the region upstream from the start codon (GENTYEX software) Downstream of the stop codon, a terminator was found and the pI was predicted to be 5.80 by the use of the N-D-AAase amino acid composition in the DNASIS software package Sequence comparison of the V paradoxus N -D-AAase protein Alignment by the BLASTP, FASTA and Swiss-Port databases using the CLUSTAL W program showed the primary structure of N-D-AAase to be similar to N-acyl-D-amino acid amidohydrolase (56.7% identity and 63.6% similarity), N-acyl-D-glutamate amidohydrolase (44.8% identity and 51.2% similarity) and N-acyl-D-asparate amidohydrolase (48.5% identity and 56.5% similarity) from Alicaligenes xylosoxydans ssp xylosoxydans A-6 and the D-aminoacylase from Alicaligenes faecalis DA1 (54.6% identity and 62.5% similarity) These results are summarized in Table The N-D-AAase protein was also similar to the genes from the complete genome sequences of Pyrococcus abyssi (42.8% identity and 53.3% similarity), Streptomyces coelicolor (35.8% identity and 42.3% similarity) and Mycobacterium tuberculosis (33.5% identity and 41.9% similarity) [46,47] Fig shows the N-D-AAase protein of V paradoxus compared to the other protein sequences in the database and using a motif search program [48] at least seven specific motifs were identified Among these motifs, all except M tuberculosis had motif 1, and while Streptomyces coelicolor did not have motif (Table and Fig 3) All other motifs were present in all the proteins The histidine residues of motifs and have already been found to be involved in the enzyme active site or structure of the N-DAAase protein [23–25] The function of the other motifs is still unknown and it will be worthwhile to further investigate N-D-AAase protein structure/function in the future Expression and purification of the N -D-AAase protein from E coli Top10 E coli harbouring pTrc2A-damA3 was cultivated in the presence of isopropyl thio-b-D-galactoside (1 mM) at 20 °C Fig Nucleotide and deduced amino acid sequence of N-D-AAase from V paradoxus The putative termination codon is indicated by asterisk Three possible )35 and )10 regions of putative promoter sequences are shown as a box Double underlining showed the potential Shine-Dalgarno sequence The putative transcription terminator is underlined Ó FEBS 2002 V paradoxus N-D-AAase gene (Eur J Biochem 269) 4873 Table Comparison of the amino acid sequence similarity of putative N-D-AAases from V paradoxus and other species The gene accession number and the strains are the same as in Fig Amino acid Accession number or strain Amino acid residues Identity (%) Similarity (%) Motif A-6-D45918 A-6-D45919 A-6-D50061 A facealis DA1 AF332548 P abyssi (strain Orsay, PAB0090) S coelicolor (cosmid 2K36, AL591857) M tuberculosis (strain H37RV, Z74024) 484 498 488 484 526 536 611 56.7 48.5 44.8 54.6 42.8 35.8 33.5 63.6 56.5 51.2 62.5 53.3 42.3 41.9 1–7 1–7 1–7 1–7 1–7 1,2,4,5,6,7 1,2,6 to avoid forming inclusion body and enzyme activity could be detected in the supernatant of the cell lysate The enzyme activity was 1.4 mg)1 higher than the E coli XL1-Blue containing pBK-damH1 (6.0 mU mg)1) From L bacterial culture, 0.18 mg protein was obtained The specific activity and the recovery of the N-D-AAase were 516.7 mg)1 and 8%, respectively (Table 2) The purified protein appeared as a single band with a few minor contaminants on SDS/PAGE with a molecular mass of 54.2 kDa (Fig 4A) The value was consistent with the predicted molecular mass Western blotting analysis gave similar results (Fig 4B) The native molecular mass of N-D-AAase protein was determined by Sephacryl HR Fig Sequence alignment of amino acid sequences of N-D-AAase from V paradoxus and other homologous proteins A-6-D45918: Alcaligenes xylosoxydans ssp xylosoxydans A-6 N-acyl-D-amino acid amidohydrolase; A-6-D45919: Alcaligenes xylosoxydans ssp xylosoxydans A-6 N-acyl-D-Asparate amidohydrolase; A-6-D50061: Alcaligenes xylosoxydans ssp xylosoxydans A-6 N-acylD-glutamate amidohydrolase; Alicaligenes faecalis-DA1: Alcaligenes faecalis DA1 N-acyl–D-amino acid amidohydrolase; V paradoxus Iso1: Variovorax paradoxus Iso1 N-acyl-D-amino acid amidohydrolase; P abyssi: Pyrococcus abyssi N-acyl-D-amino acid amidohydrolase; S coelicolor: Streptomyces coelicolor N-acyl-D-amino acid amidohydrolase; M tuberculosis: Mycobacterium tuberculosis hypothetical protein Rv2913c Sequence alignment by CLUSTAL W [37] The identical, conserved and semi-conserved amino acid residues are marked by asterisks, dots and colons, respectively The numbers represent amino acid positions Gaps were introduced to optimize the alignment The amino acid residues in the box were the motifs identified using the MOTIFSEARCH program [48] S-200 gel filtration to be 52.8 kDa and this indicated that the enzyme was monomeric IEF of the purified N-D-AAase gave a band at a pI 5.12, which was closed to predicted pI of 5.8 To confirm the protein sequence, high resolution LC/MS/MS (Finnigan LCQ) analysis was used The results gave a similarity of 100% when compared to the predicted amino acid sequence of the N-D-AAase protein Influence of temperature and pH on enzyme activity The optimal temperature for N-D-AAase was 50 °C (Fig 5A) The enzyme still had 80% activity after Ó FEBS 2002 4874 P.-H Lin et al (Eur J Biochem 269) Table Purification of the N-D-AAase from E coli pTrc2A-damA3 Enzyme activity was assayed by colorimetric assay as described in Materials and methods Steps Protein (mg) Total activity (U) Specific activity (mg)1) Recovery (%) Purification fold Crude extract Heat treatment DEAE-Toyopearl Butyl-Toyopearl Sephacryl HR FPLC-Mono Q 895 521 57 7.4 1.2 0.18 1240 1200 564 553 511 93 1.4 2.3 9.9 74.7 425.8 516.7 100 97 45 45 41 1.0 1.6 7.1 53.4 304.1 369.1 30 preincubation When the temperature was 55 °C, the treatment resulted in a 60% loss of activity (Fig 5B) Above 55 °C, activity decreased rapidly reflecting the instability of the enzyme at higher temperatures The optimal pH for enzyme activity was pH 7.5 (Fig 6A) In addition, when the N-D-AAase was preincubated at 35 °C for 30 at various different pH values, the Fig SDS/PAGE and Western blotting of N-D-AAase (A) SDS/ PAGE (10% acrylamide) M, Molecular mass standards; lane 1, crude extract; lane 2, heat treatment of crude extract; lane 3, protein after DEAE-Toyopearl purification; lane 4, protein after Butyl-Toyopearl purification; lane 5, protein after Sephacryl HR S-200 purification; lane 6, protein after FPLC-MonoQ purification step (B) Western blotting Lanes 1–6 are as described in (A) greatest stability was from pH to pH (Fig 6B) Beyond these values, in both directions, the enzyme was highly unstable Fig Optimal temperature and thermostability of N-D-AAase (A) The optimal temperature of purified enzyme Enzyme activity measurements were performed at various temperatures for 20 The highest activity was taken as 100% (B) Thermostability of purified enzyme The purified enzyme was preincubation for 30 at various temperatures Then the substrate (N-acetyl-D-methionine, 25 mM, pH 7.5) was added to the reaction and the activity was measured at 40 °C for 20 The highest activity was taken as 100% The results were the means of duplicate determinations Ó FEBS 2002 V paradoxus N-D-AAase gene (Eur J Biochem 269) 4875 Substrate specificity analysis To study the substrate specificity of the N-D-AAase protein, the activity of the enzyme against N-acyl-D- or L-amino acids and other D-amino acid derivates was determined (Table 3) The substrates analysed were a range of hydrophilic, hydrophobic and aromatic N-acyl or derivative D-amino acids that could easily be purchased from commercial source such as Sigma and Bachem The enzyme activity was 50% higher towards N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-leucine and N-chloroacetyl-D-phenylalanine than towards N-acetyl-D-valine, N-acetyl-D-phenylalanine, N-acetyl-D-tryptophan, N-acetyl-D-tyrosine and N-acetylD-asparagine However, the enzyme did not hydrolyse substrates such as N-acetyl-L-methionine and N-acetylL-leucine The results indicated the N-D-AAase protein may prefer hydrophobic amino acids such as D-methionine and D-leucine N-acetyl derivates to aromatic amino acid such as D-phenylalanine and D-tryptophan N-acetyl derivates and that there is chiral specificity A comparison of N-acetyl-D-phenylalanine and N-chloroacetyl-D-phenylalanine showed an increased activity against the latter compound and this suggests that the chloride atom of the N-chloroacetyl-D-phenylalanine substrate may promote substrate binding to the enzyme DISCUSSION Fig Optimal pH and pH stability of N-D-AAase protein (A) Optimal pH The enzyme reactions were determined at 35 °C in the following buffers (50 mM): acetate buffer (d, pH 4.0–5.6); phosphate buffer (n, pH 6.0–7.2); Tris/HCl buffer (r, pH 7.0–8.6), and Glycine/ NaOH (j, pH 9.0–10.8) The highest activity was taken as 100% (B) pH stability The purified enzyme was preincubated for 30 at 35 °C in the various buffers Substrate was then added (N-acetyl-Dmethionine, 25 mM, pH 7.5) and the enzyme activity determined for 20 at 40 °C The highest activity was taken as 100% The results were the means of duplicate determinations Influences of chelating reagents and metal ions on enzyme activity It has been reported that the enzyme activity of N-D-AAase is affected by the presence of metal ions Thus, the enzyme was treated with EDTA, EGTA, 1,10-phenanthroline and metal ions at concentrations of and 10 mM The presence of the metal ions, Fe2+, Cu2+, Zn2+, Hg2+ and Fe3+, at mM gave rise to significant inhibition of between 90% and 100% Additionally, 10 mM Ca2+, Mn2+ and Ni2+ inhibited the enzyme by 50% In contrast, significant activation or increased stability was observed with mM Co2+ and with mM Mg2+ These results indicate that the N-D-AAase protein of V paradoxus is possibly a metaldependent enzyme This study has identified a strain, Ios1, of V paradoxus, formally Alicaligenes paradoxus, belonging to the subclass b-Proteobacteria and the family Comamonadaceae At present, the Variovoras group consists of only V paradoxus [49], divided into biovar I and biovar II strains The difference between Alicaligenes and Variovorax is that the Variovorax group releases a yellow pigment into the medium, whereas Alicaligenes does not Strain Iso1 also shows nitrate reduction activity and as such is considered to be a biovar II strain [45] In addition, strain Iso1 was shown to be resistant to ampicillin (100 lgỈmL)1) and to contain a polyhydroxyalkanoates synthase gene (phaC) by PCR amplification [50] The N-D-AAase gene expressing N-acyl-D-amino acid amidohydrolase activity was cloned from V paradoxus and Table Substrate specificity of purified N-D-AAaase Relative enzyme activity was assayed by the Chirobiotic T HPLC method [44] The activity for N-acetyl-D-methionine was taken as 100% Results are the means of duplicate determinations Substrate (25 mM) Relative activity (%) N-acetyl-D-methioine N-acetyl-D-alanine N-acetyl-D-valine N-acetyl-D-leucine N-acetyl-D-phenylalanine N-acetyl-D-tryptophan N-acetyl-D-tyrosine N-chloracetyl-D-phenylalanine N-acetyl-D-asparagine N-acetyl-L-methionine N-acetyl-L-leucine 100 53 18 84 24 201 19 0 ± ± ± ± ± ± ± ± ± 4.1 5.2 1.3 2.2 2.2 0.2 0.3 2.2 2.2 Ó FEBS 2002 4876 P.-H Lin et al (Eur J Biochem 269) its nucleotide sequence determined Upstream of the ORF, three possible promoter regions were identified (Fig 2) These were the )35 regions TTGGCA )192 to )187 bp, TGGTCA )152 to )147 bp, CTGAGC )99 to )104 bp and the )10 regions TATGGT )165 to )160 bp, GACACT )131 to )126 bp and TACATC )73 to )68 bp The plasmid pBK-damD4 showed enzyme activity indicating that one or more of these promoter regions could be recognized by an E coli RNA polymerase Interestingly, another ORF was found on the complementary strand of the N-D-AAase gene: its gene length was 1149 bp encoding 382 amino acid residues; however, this showed no significant similarity to any gene in the GenBank database When N-D-AAase was expressed in E coli originally, active soluble protein production could not be obtained at 37 °C even at an isopropyl thio-b-D-galactoside concentration lower than mM When the temperature was downshifted to 20 °C at mM isopropyl thio-b-D-galactoside induction, high soluble protein activity was detected This indicated that the lower temperature might help the cells to fold the active protein correctly Some conserved motifs could be identified when N-D-AAase protein sequence was compared with other similar proteins with greater than 50% similarity (Table and Fig 3) Among these motifs, the first histidine residue of motif (DXHXH) is considered to be involved in the catalytic site of the enzyme and the second histidine residue may play a role in maintaining the enzyme structure Additionally, the first histidine residue of motif is considered to be involved in metallic ion binding and enzyme catalytic function [23] The function of the N-D-AAase protein in the bacterium is not very clear, but recently one study of V paradoxus has suggested that the aminoacylase may be used to hydrolyse N-butanoyl-homoserine lactones (C4-HSL) to produce HSL and fatty acids, which are then used as the sole energy and nitrogen sources [51] The acyl-HSL signalling molecules may be biologically inactivated by specific soil bacteria Here, the N-D-AAase from V paradoxus may possibly play a role in the degradation of acyl-HSL molecules and this needs to be tested in the future According to the results of the peptide sequencing determined by LC/MS/MS analysis, some methionine residues seemed to be modified because there was a molecular weight increase of 16 These methionine residues were Met39, Met171, Met254, Met273 and Met352 It is known that the common sites of oxidation in proteins are histidine, lysine, proline, cysteine, arginine and methionine residues [52] Methionine oxidation can be caused by protein damage or aging by endogenous or oxidizing agents [53,54] and maybe the reason why the N-D-AAase enzyme of V paradoxus purified from E coli is unstable when the enzyme is stored at °C Under these conditions, enzyme activity decreased very rapidly over a few days Although some reports have shown that methionine oxidation has no influence on protein function [55,56], others have shown that 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Alcaligenes xylosoxydans ssp xylosoxydans A- 6 N- acylD-glutamate amidohydrolase; Alicaligenes faecalis-DA1: Alcaligenes faecalis DA1 N- acyl? ?D-amino acid amidohydrolase; V paradoxus Iso1: Variovorax. .. A- 6-D45918: Alcaligenes xylosoxydans ssp xylosoxydans A- 6 N- acyl -D-amino acid amidohydrolase; A- 6-D45919: Alcaligenes xylosoxydans ssp xylosoxydans A- 6 N- acyl-D-Asparate amidohydrolase; A- 6-D50061: Alcaligenes