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Keywords:

  • Achromobacter xylosoxidans;
  • Amidase;
  • Amidase signature family;
  • Ana;
  • GC content;
  • Wide-spectrum

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

Amidases are very important enzymes for industrial biocatalysis. We scored a novel amidase by screening the Achromobacter xylosoxidans gene library with cephalosporin analogous amides. The gene coding for the enzyme, designated ana, was cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis of ana showed it to be an amidase signature family member. Interestingly, we noted that almost all Ana homologous amidases are from human pathogens responsible for chronic lung infections. Knowing the genetic context of Ana and its homologous amidases, we suggest that they could be a part of transposon structure. Ana can efficiently hydrolyze a series of cephalosporin analogous amides, including amides with an aninine, p-nitro-aninine, and β-naphthylamine moiety, while cephalosporin could not serve as its substrate.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

Amidases are a class of enzymes that convert amides to acids and have potential value in the development of commercial bioprocesses for the production of useful chemicals [1]. The amidase signature family (AS) is defined by a highly conserved stretch of approximately 130 serine- and glycine-rich amino acids containing three invariant residues responsible for catalysis. Until now, the crystal structures of three members of the amidase signature family, including peptide amidase (Pam) from Stenotrophomonas maltophilia[2], malonamidase E2 (MAE2) from Bradyrhizobium japonicum[3], and fatty acid amide hydrolase (FAAH) from Rattus norvegicus[4], were determined. The proteins of the amidase signature family are classified as non-classical serine hydrolases. The catalytic triad of this enzyme family is not the classical Ser-His-Asp, but a novel triad, Ser-cis Ser-Lys [2–4]. AS enzymes represent a large family of non-classical serine hydrolase, which is widespread in nature, exhibits a variety of biological functions, and contains more than 200 proteins from over 90 different organisms including bacteria, archaea and eukarya [2]. The biological substrate of all known AS enzymes are amides. AS family members are evolutionarily distinct but have diverged to acquire a wide spectrum of individual substrate specificities, while maintaining a core structure that supports the catalytic function of the unique triad [3].

Achromobacter xylosoxidans is a rare but important cause of bacteremia in immunocompromised patients, and strains are usually multiply resistant to antimicrobial therapy [5]. We characterized novel penicillin G acylase (PGA650) and putative cephalosporin acylase [5,6]. A. xylosoxidans has also been described as having PVA activity and an unspecific enzyme synthesizing cephalexin [7]. It is the probably the only strain yet characterized that harbors three kinds of β-lactam acylases together. During our attempts to clone a putative CPC acylase in A. xylosoxidans, we scored one novel amidase (Ana) with deacylation activity towards several cephalosporin structural analogs. In this manuscript, we describe the cloning and characterization of Ana. This enzyme was found to belong to the amidase signature family, showing significant sequence similarity to putative amidases from several lung pathogens (such as Bordetella bronchiseptica, Bordetella pertussis and Mycobacterium tuberculosis H37RV). Expression, purification, genetic context, kinetic characterization and substrate profile of Ana are reported.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

2.1Bacterial strains and culture conditions

The A. xylosoxidans strain used in this study is from our lab storage. Escherichia coli strains DH5a and JM109 (laboratory stock) were used for routine cloning procedures. The plasmids pBC-SK and pBluescript KS (Stratagene) were used as cloning vectors. Overproduction of Ana was performed with E. coli BL21 (DE3) (Novagen). The E. coli and A. xylosoxidans strains were routinely grown and maintained in LB medium with appropriate antibiotic selection.

2.2Chemicals

We synthesized seven model compounds: 5-adipoylamido-2-nitro-benzoic acid (Ad-NABA); 5-glutaryl-amido-2-nitrobenzoicacid (Gl-NABA); glutaryl-naphthylamine (GL-Nap); glutaryl-aniline (Gl-Aniline); glutaryl-p-nitroaniline (Gl-NA); adipoyl-p-nitroaniline (Ad-NA); and 6-nitro-3-phenylacetamino-benzoic acid (NIPAB), which mimick the β-lactam moiety of cephalosporin and were utilized as substrates for the rapid screening of microorganisms harboring cephalosporin acylases [8].

2.3Gene cloning and sequencing

The construction of the genomic DNA library of A. xylosoxidans was described previously [5]. Recombinant clones harboring the ana gene were selected for the expression of Ana amidase on the basis of formation of yellow halos on Ad-NABA (1 mg/ml) plates. The positive clone (JM109/pAna) was obtained and the 3.4-kb insertion was sequenced using the dideoxy-chain termination method. To enhance Ana expression levels in E. coli, the recombinant plasmid pAna was constructed. In construction of pAna, the ANA-encoding gene ana was amplified by PCR with two oligonucleotides, Ana L (5-GCCATATGATGGGCACGGCTGACCGA-3) and Ana R (5-CGGAATTCGGCCTATCTGGCGATGG-3) (underlined sequences are Nde I and Eco RI recognition sites, respectively). The resulting 1.5-kb PCR fragment was cloned into the Eco RV site of pBluescript II KS (+) to generate pKSana. pKSana was doubly digested with Eco RI and Nde I, and the released insert was cloned into pET28b(+) (Novagen) to generate pAna. pAna was introduced into E. coli strain BL21(DE3) (Novagen) to allow expression of Ana under the control of the T7 lac promoter.

2.4Protein expression and purification

The E. coli strain BL21 (DE3) cells containing pAna were harvested by centrifugation at 4000 rpm for 20 min in a Beckman J2-HS instrument and resuspended in 10 ml of lysis buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole) after induction with 0.8 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 3–4 h at 26°C. The sample was lysed by sonication on ice in an ultrasonic homogenizer (Cole–Parmer Instrument, Chicago, IL). The lysate was then centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant containing soluble cellular protein was loaded onto an immobilized metal affinity chromatography (QIAGEN). The column containing 2 mL of Ni–NTA affinity resin was equilibrated with 10 column volumes of lysis buffer in advance. The column then was washed with 5 column volumes of wash buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 50 mM imidazole). The bound Ana recombinant protein was eluted with 3 column volumes of elution buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 250 mM imidazole). The active fractions were pooled, dialyzed and analyzed by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS–PAGE). All purification steps were carried out at 4°C unless stated. The purified Ana protein was analyzed by SDS–PAGE.

2.5Sequence analysis

Amino acid similarity and identity percentages were determined by ClustalW multiple sequence alignment [9]. The World Wide Web Prediction Server SignalP V1.1 (http://www.cbs.dtu.dk/services/SignalP/) [10] was used to predict the presence of signal peptide cleavage sites in amino acid sequences. Phylogenetic relationships and evolutionary distances were analyzed by using the PROTDIST, FITCH, DRAWGRAM and DRAWTREE routines of the PHYLIP 3.6a program, which is available at the Pasteur Institute server (http://bioweb.pasteur.fr/seqAnal/phylogeny/phylipuk.html) [11].

2.6Enzyme assay and characterization

Ad-NABA was used as the substrate for routine assays. The enzymatic hydrolysis of Ad-NABA, Gl-NABA and Gl-NA was monitored at 37°C for 5 min in a solution containing a 100 mM potassium phosphate buffer (pH 7.5), 0.5 mg/ml Ad-NABA (Gl-NABA or Gl-NA), and an appropriate amount of the enzyme. The reaction was started by the addition of the substrate to the pre-warmed reaction mixture and stopped by adding an equal volume of a mixture (2:1) of acetic acid (20% in water) and 50 mM NaOH. The increase in the absorbance at 415 nm, indicating the formation of 2-nitro-5-amido benzoic acid (NABA) or nitroaniline, was recorded. One unit of enzyme activity was defined as the amount of enzyme that produced 1 mmol of NABA per min at 37°C and pH 7.5. Amidase activities toward GL-Nap, GL-Aniline and CPC of Ana enzyme was determined by the pDAB colorimetric method [12].

The optimal temperature for activity was determined in a range of 25–55°C by measuring Ad-NABA hydrolysis. The effect of pH on the activity of the enzyme was determined with Ad-NABA as the substrate at 37°C in 50 mM phosphate buffer (pH 6–8) or 50 mM Na2HPO4–NaOH (pH 9–10).

2.7Nucleotide sequence Accession Numbers

The nucleotide and the protein sequences of Ana reported here have been submitted to the GenBank database under Accession Nos. AY515701 and AAS87173, respectively.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

3.1Cloning and sequencing of Ana gene from A. xylosoxidans

A positive clone harboring Ana named JM109/pANA was obtained through screening the A. xylosoxidans genomic DNA library with an Ad-NABA substrate. The clone carries a 3.4-kb insert which was sequenced on both strands. (GenBank Accession No. AY515701) to reveal three open reading frames. The ana ORF contained the complete ana gene encoding a polypeptide of 509 amino acids. Sequence analysis revealed a potential ATG start codon of Ana at nucleotide position 1328, proceeded by a putative Shine Dalgarno sequence (GGAG at 1314–1347 bp position). Two further open reading frames, labeled cat and acd, were found just downstream and upstream of the coding region of Ana. The cat shows strong similarity (more than 99% identity) to chloramphenicol acetyltransferase [13]. The other ORF (acd) lies downstream from ana extending to the end of the sequenced fragment and in the same strand. The acd showed significant similarity (more than 60% identity) to genes encoding well-characterized acyl-CoA dehydrogenases [14].

The average GC content of the Ana coding region was 71% and its codon usage preferred G or C in the wobble positions. The 3.4 kb sequenced region showed strikingly inconsistent GC content and the highest GC content region attains 90.2%, while the lowest is only 28%. The GC content of the cat gene was significantly lower than the mean GC values of the total sequenced region. It is known that the cat gene is often associated with a transposable element. Indeed, we found that Ana homologous amidases are invariable flanked with transposases (GenBank Accession No. BX640444 and BX640417), suggesting that they could be part of a transposon structure [15]. These observations suggested that the ana gene might be part of a transposon element and a relatively recent acquisition for A. xylosoxidans.

3.2Amino acid sequence analyses of Ana

A BLAST analysis of the encoded Ana protein sequence revealed homologies to a variety of amidases, all belonging to the amidase signature superfamily. The amidase protein sequence was compared to other homologous amidase sequences using the ClustalW program (data not shown) and showed typical amidase signature superfamily amidase organization. As can be observed, the Ana sequence presented significant similarity to known amidase signature superfamily counterparts [2–4], especially regarding the catalytically important residues that are highly conserved. A phylogenetic tree of Ana and its homologous amidases was constructed by the PHYLIP 3.6a program (Fig. 1). It showed that Ana from A. xylosoxidans is phylogenetically closely related to the putative amidases from B. bronchiseptica, B. pertussis, Mycobacterium leprae, Rhodobacter sphaeroides, Pseudomonas aeruginosa and S. maltophilia. This is consistent with the relative relationship for these taxa. Interestingly, we noted that almost all these strains are gram-negative non-fermenting bacilli and opportunistic human pathogens that cause chronic lung infection.

image

Figure 1. Phylogenetic tree of Ana and its homologous amidases based on the enzymes’ amino acid sequences. The tree was constructed using the ClustalW and PHYLIP 3.6a program by the neighbor-joining method as described in Section 2 (Acinetobacter sp. EstA8 [16]). Bootstrap value with neighbor-joining search is also shown. The scale bar represents 10 amino acid residue substitutions per 100 amino acid residues.

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3.3Heterologous expression of Ana in E. coli

We successfully cloned ana in the highly efficient overexpression vector pET28b(+) under the control of the T7 lac promoter. Generated pAna was introduced into E. coli strain BL21 (DE3) (Novagen), which is utilized for efficient expression of the Ana protein. Amidase production was monitored by measuring the Ad-NABA specific activity. The induction of the amidase activity in the recombinant E. coli cultures was optimized using different amounts of IPTG, revealing 0.8 mM to be the optimal concentration (data not shown). The optimal growth temperature for Ana expression in E. coli was about 26°C (data not shown). We found that higher cultivation temperatures, such as 37°C, led to lower specific activity of Ana, while lower cultivation temperatures, such as 16 or 20°C, presented poor biomass for further protein isolation.

3.4Purification of Ana from recombinant E. coli

The Ana protein from the crude extract of E. coli BL21 (DE3) harboring pAna was purified to more than 99% homogeneity by Ni–NTA agarose affinity chromatography (Fig. 2). The purified enzyme showed one band on SDS–PAGE.

image

Figure 2. SDS–PAGE analysis of Ana purified from recombinant E. coli BL21 (DE3)/pAna. Gels were stained with 0.025% Coomassie brilliant blue R-250 after electrophoresis. The molecular masses of marker proteins (left) are indicated in kilodaltons. The band corresponding to Ana is indicated on the right.

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The biochemical characteristics (molecular mass, enzymatic activity, and activity dependency on temperature and pH) were determined for both purified recombinant Ana and the native enzyme, which were very similar.

3.5Characterization and kinetic properties of recombinant Ana

The specific activity of the purified Ana enzyme towards Ad-NABA and Gl-NABA was about 4.67 and 3.5 units/mg, respectively (Table 1). The Km values for Ad-NABA and Gl-NABA were 0.315 and 0.958 mM, respectively, which suggested that Ana has a higher affinity to the moiety of adipoyl group than that of glutaryl group. The temperature and pH dependence of Ana analysis suggested that the optimal temperature for Ana activity was about 44°C (Fig. 3(a)) and the optimal pH for Ana hydrolytic activity was close to 8.0 (Fig. 3(b)). The activity drastically dropped when the reaction was carried out at temperature higher than 50°C or at a pH below 6.0.

Table 1.  Reactions catalyzed by Ana and enzymatic parameters of purified A. xylosoxidans Anaa
  1. aKinetic parameters of Ana are mean values of triplicate measurements. All data were measured at 37°C and at pH 7.5.

inline image
SubstrateRKm (mM)Vmax (IU/mg)
Ad-NABAHOOC–(CH2)4–(adipyl)0.315 ± 0.012150 ± 8
Gl-NABAHOOC–(CH2)3–(glutaryl)0.958 ± 0.043130 ± 5
image

Figure 3. Effect of temperature and pH on the activity of recombinant purified Ana. (a) Temperature–activity profile of Ana. The enzyme activity was assayed at each temperature in 50 mM phosphate buffer (pH 8) by measuring Ad-NABA hydrolysis. (b) pH–activity profile of Ana. The effect of pH on the activity of the enzyme was determined at 37°C in 50 mM phosphate buffer (pH 6–8) and 50 mM Na2HPO4/NaOH (pH 9–10) for the Ad-NABA substrate. 100% activity corresponds to the enzymatic activity of the optimum temperature (44°C) and pH (8.0), respectively.

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3.6Substrate specificity of Ana

The activities of Ana towards several cephalosporin analogous amide compounds and cephalosporin were tested (Table 2). Ana also showed a weak but significant activity toward amides with an aninine and β-naphthylamine moiety such as glutaryl-aninine and glutaryl-β-naphthylamine. This wide substrate specificity profile is likely to contribute to hydrolysis of a vast number of small amides transported into the cell. Although Ana was found to have deacylation activity on a series of cephalosporin derivatives, cephalosporin itself could not serve as its substrate.

Table 2.  Acylase activity of A. xylosoxidans Ana on various amides tested by colorimetric assaya
SubstrateStructureSpecific activity (unit/mg)Relative activity
  1. aThe response level based on staining intensity and ranging from – (colorless) to +++ (very intense yellow solution), were visually determined after 30 min of reaction. +++, strong activity; ++, active; -, no activity and nd, not determined.

Adipoyl-nitro-5-amino benzoic acid (Ad-NABA)inline image4.67 ± 0.11+++
Glutaryl-nitro-5-amino benzoic acid (Gl-NABA)inline image3.51 ± 0.09+++
Adipoyl-p-nitro-aninine (Ad-NA)inline imagend+++
Glutaryl-p-nitro-aninine (Gl-NA)inline image2.29 ± 0.05+++
Glutaryl-β-naphthylamine (Gl-Nap)inline imagend++
Glutaryl-aniline (Gl-Aniline)inline imagend++
Cephalosporin C (CPC)inline image0

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

This work was supported by the National High Technology Development Program (2001AA235081) and the National Basic Research Program (2003CB716000) of China. We appreciate the kind help of Tianfan Cheng with the phylogenetic analysis.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References
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