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Adenosine deaminase from Streptomyces coelicolor: Recombinant expression, purification and characterization
Protein Expression and Purification 78 (2011) 167–173
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Adenosine deaminase from Streptomyces coelicolor: Recombinant expression, purification and characterization Somchai Pornbanlualap ⇑, Pornchanok Chalopagorn Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand
a r t i c l e
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Article history: Received 11 January 2011 and in revised form 28 March 2011 Available online 12 April 2011 Keywords: Streptomyces coelicolor Adenosine deaminase Adenine deaminase Transition state 20 -Deoxycoformycin Divergent evolution
a b s t r a c t The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr = 48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s1at 30 °C. Since adenine is deaminated 103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-b-D-arabinofuranosyl-adenine (ara-A), 50 -AMP, 50 -ADP and 50 -ATP, are not substrates for the enzyme. Coformycin and 20 deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common a/b barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. Ó 2011 Elsevier Inc. All rights reserved.
Introduction Adenosine deaminase (ADA, EC 3.5.4.4) catalyzes the irreversible deamination of adenosine to inosine and ammonia. This enzyme has been found in a wide variety of microorganisms, plants, invertebrates and mammals. It is an indispensable enzyme in purine metabolism. In humans, this enzyme is required for B and T-cell development and plays a central role in the maintenance of a competent immune system [1]. Genetic deficiency of ADA results in a disease known as severe combined immunodeficiency disease (SCID), which is characterized by a lack of T- and B-lymphocytes. Children with this disease have no detectable lymphocytes and are subject to recurrent infections [2]. The crystal structure of murine ADA shows that this protein is a zinc metalloenzyme that displays a typical ab-barrel fold, with eight central b-strands and eight peripheral a-helices [3]. In addition to this central motif, the structure contains five additional a-helices. The catalytic mechanism of deamination has been ⇑ Corresponding author. Fax: +66 2 561 4627. E-mail address: [email protected] (S. Pornbanlualap). 1046-5928/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2011.04.003
suggested to proceed by the direct water attack on the C-6 position of the purine ring, resulting in the formation of a tetrahedral intermediate [4] (Fig. 1). Thus, 6-hydroxyl-1,6-dihydropurine riboside (HDPR) and the naturally occurring nucleoside antibiotic, 20 -deoxycoformycin, whose structures mimic the hypothetical tetrahedral transition state intermediate, bind tightly to the enzyme, with Ki of 1013 and 1012 M, respectively. Adenine deaminase (ADE) and AMP deaminase are enzymes mechanistically related to ADA. ADE is also involved in the purine salvage pathway by deamination of adenine to hypoxanthine and ammonia. AMP deaminase catalyzes the formation of IMP and ammonia from AMP and is involved in regulating adenine nucleotide levels in eukaryotes [5]. Recent phylogenic analysis of amino acid sequences of various gene products suggests that ADAs, ADEs, AMP deaminases, adenosine deaminase-like (ADAL), and adenosine deaminase-related growth factors (ADGF) are a group of proteins that belong to the adenyl-deaminase family [6]. Members of this family share a novel motif consisting of methionine (or iso/leucine), proline, lysine and glycine (MPKG). In addition to this novel motif, the catalytically active residues are highly conserved in all five protein subfamilies.
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Fig. 1. (A) Reaction coordinates for deamination of adenosine to inosine catalyzed by adenosine deaminase. The reaction occurs by direct water attack on C6 of adenosine to generate a tetrahedral intermediate. The relative energy along the reaction coordinates is arbitrary with the highest energetic point as the transition state. As substrate is transformed into the transition state and changes in the [C6–Ohydroxyl], [C6–N6], [C1–N1] and [N1–H] bond lengths occurs. Modified from Luo et al. [22]. (B) Structure of competitive inhibitor, 6R-hydroxyl-1,6-dihydropurine riboside (HDPR), formed by covalent hydration of purine riboside. (C) Structure of 8-(R)-coformycin, a nucleoside antibiotic and a transition state inhibitor. The sugar substituent R in coformycin is ribose and in 20 -deoxycformycin is 20 -deoxyribose.
Plasmid construction
Streptomycetes are a group of filamentous Gram-positive, soilinhabiting bacteria which have captured enormous screening interest because of their ability to produce and secrete a variety of antibiotics and extracellular proteins [7]. Among the diverse antibiotics produced by Streptomycetes are the nucleoside antibiotics. The sequence of the genome of Streptomyces coelicolor A3(2) has been predicted to contain genes encoding more than 7500 proteins, many of which have yet to be studied and defined [8]. SCO4901 is one of these genes that had been annotated in data bases as a ‘‘putative’’ adenosine deaminase. However, since ADA and ADE proteins have been shown to have approximately the same size, exhibit overall sequence similarity, contain similar folding topology and share the same catalytic machinery, it is often difficult to unambiguously distinguish one from the other. Characterization of the ADE sequences from Aspergillus nidulans, Saccharomyces cerevisiae and Schilzosaccharomyces pombe shows that these proteins are more closely related to the biochemically characterized ADAs rather than Bacillus subtilis and Escherichia coli ADEs. Based on sequence alignment of ascomycete ADEs and the characterized ADAs, a structural rule has been proposed to distinguish whether a protein is an ADA or an ADE [9]. Asp19, Ser103, Ala183, and Gly184 (numbered according to murine ADA) have been proposed to be characteristic of ADA, whereas Glu, Asp, Asp or Ser, and Ser at the same position are characteristic of ADE. Although Streptomycetes are the major producers of nucleoside antibiotics and many of nucleoside antibiotics exhibit inhibitory effects on purine salvaging enzymes, few enzymes of Streptomycetes origin have been biochemically characterized. Therefore, this report describes the expression, characterization of substrate specificity and inhibition of adenosine deaminase from S. coelicolor (ScADA) by transition state analogs.
Genomic DNA was purified from S. coelicolor cells grown in yeast extract-malt extract (YEME) containing 34% sucrose, 1% glucose and 0.5% MgCl2 at 30 °C as described by Hopwood et al. [10]. The concentration of genomic DNA was measured in a UV spectrophotometer (at 260 nm). The size of genomic DNA was analyzed on 1% agarose gels. The forward primer (ADA-1: 50 -TTTAAGCTTCATATG ACGAGCCGAAGCACCGAG-30 , where the underline indicates the added HindIII and NdeI linkers) and the reverse primer (ADA2: 50 -TTTAGATCTTCAGCCGTCG GATTCCGAAGA-30 , where the underline indicates the added BglII linker) were designed according to the putative adenosine deaminase gene sequence from S. coelicolor (Genebank Accession No. SCO4901). The complete coding region of the gene was amplified by PCR with ADA-1 and ADA-2 primers, using chromosomal DNA prepared from S. coelicolor as the template and Taq DNA polymerase. PCR was performed as follows: initial denaturing at 93 °C for 5 min; 30 cycles of denaturation at 93 °C for 1 min, annealing at 68 °C for 1 min, and additional extension at 72 °C for 1.5 min, and additional extension at 72 °C for 10 min. The PCR product was gel purified with a PCR purification kit (Qiagen) and ligated into the pGEM-T vector (Promega). After transformation into competent E. coli JM109, recombinant plasmid, pGEM-ada, was purified from positive clones by the alkali method and sequenced to confirm that no mutations were introduced by the PCR amplification step. After a double digestion of pGEM-ada with NdeI and BglII, the 1.2 kb fragment was gel purified and ligated into pET-15b, which had been previously double digested with NdeI and BamHI. The ligation mixture was transformed into E. coli BL21 (DE3) and selected on LB containing 60 lg/ml ampicillin.
Materials and methods
Protein purification
Materials
For purification of adenosine deaminase, 5 ml of E. coli BL21 (DE3) carrying pET-ada from overnight grown culture in LB supplemented with 60 lg/ml ampicillin was transferred to one liter of LB plus ampicillin. The culture was allowed to grow to an OD600 nm of 0.5 and induced by addition of lactose at a final concentration of 1 mM, since lactose had been shown to be as an effective inducer as IPTG [11]. After five additional hours of growth, cells were harvested by centrifugation at 10,000g at 4 °C, suspended in lysis buffer [20 mM Tris–HCl (pH 8.0), 50 mM KCl, 1 mM EDTA, 0.5% Tween
Adenosine, 20 -deoxyadenosine, adenine, 9-b-D-arabinofuranosyladenine (ara-A), and IPTG were from Sigma/Aldrich. 20 -Deoxycoformycin and coformycin were provided by Dr. Robert J. Suhadolnik, Temple University School of Medicine (USA). Nickelagarose was from Qiagen. S. coelicolor A3(2) was a generous gift from Dr. Keith Chater, John Innes Institute (UK). E. coli strain BL21 (DE3) and pET-15b were from Novagen.
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20 and 1 mM PMSF] and disrupted by sonication. The sonicator was programmed to provide 15 s pulses with a 10 s pause for a total period of 30 min. After centrifugation at 12,000g for 30 min, the enzyme in the supernatant was precipitated by addition of ammonium sulfate at 80% saturation. Precipitated proteins were collected by centrifugation at 10,000g, dissolved in 4 ml of lysis buffer and dialyzed four times against 3 l of buffer containing 50 mM potassium phosphate (pH 7.5). The dialyzed protein was added onto Ni2+-NTA resin (1 ml bed volume), which had been previously equilibrated with buffer B [20 mM Tris–HCl (pH 8), 500 mM KCl, and 0.1% Triton X-100]. After washing the column with 4 ml of buffer B, the polyhistidine-tagged enzyme was eluted by washing with buffer B containing 20, 100 and 250 mM imidazole, respectively. The enzyme eluted from column was analyzed on 10% SDS–PAGE. Assay for adenosine deaminase activity The activity of adenosine deaminase was assayed spectrophotometrically by measuring the decrease in absorbance at 265 nm on conversion of adenosine to inosine with a Shimazu spectrophotometer. One milliliter reactions containing 0.04–0.08 lmole of adenosine in 50 mM potassium phosphate were incubated for 1 min in a cuvette prior to the addition of enzyme. The amount of product formed was calculated using a molar extinction coefficient of 8.4 mM1 [12]. The same wavelength and molar extinction coefficients were used for determination of the Km and Vmax for ara-A, 20 -deoxyadenosine, adenine, AMP, ADP, and ATP. One unit of enzyme activity is defined as the amount of enzyme needed to convert 1 lmole of substrate to product at 37 °C in one minute. Protein concentration was determined by the Bradford method, using bovine serum albumin as standard. Analysis of kinetic data Kinetic data were fitted to the appropriate equations using the Sigma Plot program. The initial velocity (v) obtained by varying the concentration of substrate was fitted to the equation: v = Vmax [S]/Km + [S], where Vmax is the maximum velocity, [S] is substrate concentration, and Km is the Michaelis constant. Kinetic data obtained from competitive inhibition were fitted to the equation: v = Vmax [S]/Km (1+[I]/Ki) + [S], where [I] is the concentration of inhibitor and Ki is the inhibition constant. Molecular weight estimation The elution position of active ADA relative to protein of known molecular weight was determined by size exclusion chromatography, using HiPrep 16/60 Sephacryl S-200HR column (Pharmacia). A solution (0.5 ml) containing 0.4 mg/ml of ADA, 10 units of pyruvate kinase (237 kDa), 3 mg/ml conalbumin (75 kDa), 4 mg/ml ovalbumin (43 kDa), 50 mM KH2PO4 (pH 7.0) and 0.15 M NaCl was applied to a column which had been equilibrated with the same buffer but lacking the enzymes. The flow rate was 1 ml/minute and the elution profiles were monitored at 280 nm by UV absorption. The elution position of ADA was detected by the deamination assay described above. Metal analysis Purified protein samples were analyzed for zinc content with Varian atomic absorption spectrophotometer model AA280 FS at 213.8 nm. Protein was quantified using the predicted molar extinction coefficient (at 44,265 M1 cm1 at 280 nm) and by the Bradford method, using bovine serum albumin as standard.
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Results Cloning of the putative adenosine deaminase gene Using the ADA-1 and ADA-2 primers, PCR reactions resulted in a single band of the correct length 1.2 kb. The nucleotide sequence of gel-purified DNA was confirmed as a putative adenosine deaminase from S. coelicolor by sequencing before it was cloned into pET-15b. The resultant plasmid, pET-ada, was expected to express a protein of 396 amino acids with 20 additional amino acids, including a polyhistidine tag at the N-terminus and a predicted molecular mass of 45,987 Da. The crystal structure of murine ADA (MuADA) shows that the enzyme folds as an eight stranded parallel ab barrel [3]. Since the active site of proteins with (a/b)8 barrel domain is located at the COOH-terminal face of b barrel, addition of polyhistidine at the NH2-terminus of enzyme was expected to have little or no effect on the catalytic activity. Purification of the adenosine deaminase A major band with molecular weight of approximately 48 KDa was observed after five hours of induction with 1 mM lactose (Fig. 2A). The purification of recombinant S. coelicolor adenosine deaminase (ScADA) from one liter of E. coli BL21(DE3) carrying the pET-ada cell culture resulted in a total of 16.5 mg protein with the specific activity of 2.07 lmole/min/mg (Table 1) and 85% homogeneity as estimated from denaturing polyacrylamide gel electrophoresis (Fig. 2A). When the molecular weight of the enzyme was estimated by gel-exclusion chromatography using proteins of known masses as standards, ScADA eluted at an apparent molecular weight of 182,000 (Fig. 2B). These values are consistent with the active enzyme being composed of four subunits with identical molecular weight. This is in contrast to murine, human and plasmodial ADAs, where the enzymes are active as monomers [13–15]. Substrate/inhibitor specificity and zinc requirement The kinetic behavior of the recombinant enzyme with adenosine and 20 -deoxyadenosine may be reasonably well described by the simple Michaelis–Menten model. The Km, Ki, and Vmax values of purified enzyme for substrates/inhibitors are shown in Table 2. Substrate specificity studies show that adenosine is the best substrate with Vmax of 15.4 (lmole/min/mg) and turnover number (kcat) of 11.5 s1. Substitution of the hydroxyl group at the 20 -position with hydrogen (i.e., 20 -deoxyadenosine) is moderately tolerated, resulting in a decrease of relative Vmax to 17.5% as compared to adenosine and more than a 15-fold increase in Km. Removal of the ribosyl moiety from adenosine (i.e., adenine) or inversion of the hydroxyl group at the 20 -position (i.e., ara-A) results in almost complete loss of activity as substrate. (Vmax < 0.06%). Addition of the phosphate group to the 50 -position of the ribosyl moiety (i.e., 50 -AMP, 50 -ADP, and 50 -ATP) also results in complete loss of activity as substrate. Since it is not deaminated by the enzyme, adenine was tested as an inhibitor of the enzyme. The result shows that adenine is a poor inhibitor of the enzyme with a Ki > 1 mM. Aspergillus oryzae and calf intestinal ADAs bind to adenosine and adenine with similar Km, although the former is deaminated 103–104 faster than the latter [16]. Coformycin and 20 -deoxycoformycin, whose structures mimic the transition state of the deamination reaction, are potent competitive inhibitors of the enzyme, with Ki of 2.5 1010 and 3.4 109 M, respectively (Fig. 3). Substrate specificity and inhibitor studies showed that the presence of the hydroxyl group at the C-20 position of the ribosyl moiety appears to contribute to tight
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Fig. 2. Analysis of the purified adenosine deaminase by polyacrylamide gel electrophoresis and gel exclusion chromatography. Panel A: lane 1, standard protein markers; lane 2, cell-free extract before induction; lane 3, cell-free extract after induced with lactose; lane 4, 5 lg of protein isolated following Ni–NTA chromatography step. Panel B: Elution position of native adenosine deaminase relative to proteins of known molecular weight (see Materials and methods section).
Table 1 Summary of the purification of S. coelicolor adenosine deaminase. Step
Total protein (mg)
Total unit (U)
Specific activity (l mole/min/mg)
Fold purification
Recovery (%)
Cell-free extract 0–80% (NH4)2SO4 Ni2+-NTA affinity
120 38 16.5
84.8 36.5 34.2
0.35 0.96 2.07
1.0 2.7 5.9
100 43 40
Table 2 Comparison of Km, Ki, and Vmax values for the interaction of adenosine analogs with adenosine deaminase from S. coelicolor. Substrate or inhibitor Substrates Adenosine 20 -Deoxyadenosine Ara-A Adenine 50 -AMP 50 -ADP 50 -ATP Inhibitors Coformycin 20 -Deoxycoformycin Adenine
Km (lM)
Ki (lM)
29 560
Vmax (l mole/min/mg)
Vmax (%)
15.4 2.7 <0.011 <0.011 ND ND ND
100.0 17.5 <0.06 <0.06
0.00025 0.0034 >1000
ND – not detectable. 1 The deamination was too slow to allow determination of Km.
binding of the substrate or inhibitor to the enzyme, since ribonucleosides (adenosine and coformycin) bind tighter to the enzyme than their deoxyribonucleoside counterparts (20 -deoxyadenosine and 20 -deoxycoformycin). In contrast, ADA isolated from other sources has been reported to bind slightly tighter to 20 -deoxycoformycin than coformycin. No slow onset of inhibition of ScADA by coformycin or 20 -deoxycoformycin was observed under our experimental conditions. The purified ScADA requires zinc for catalytic activity. In the absence of 1,10-phenanthroline, ScADA retained virtually 100% of activity for 60 min (data not shown). When the enzyme was incubated 0.7 mM of 1,10-phenanthroline, the enzyme lost approximately 90% of its catalytic activity after 60 min of incubation. Analysis of the metal content of purified enzyme by atomic absorption spectroscopy shows that the enzyme contains approximately 0.7 mole of tightly bound zinc/mole of subunits (Table 4). The less than the expected 1:1 ratio of zinc/ subunit of enzyme is perhaps due to insufficient amount of zinc presence in the growth medium needed to be incorporated into the expressed enzyme upon induction with lactose.
Fig. 3. The effect of 20 -deoxycoformycin (20 -dCF) on the initial rate of product formation by adenosine deaminase. Inhibition of ADA by 20 -dCF was measured in 50 mM potassium phosphate buffer, pH 7.0. The initial rates were measured by the decrease in optical density at 265 nm. The data points were fitted to an equation for competitive inhibition.
Discussion The purposes of this study were to (i) determine whether SCO4901 encodes for ADA or ADE, (ii) purify the recombinant protein and determine its subunit structure, (iii) characterize substrate and inhibitor specificity of the enzyme and (iv) elucidate the evolutionally relationship between ADA and ADE. To this date, no ADA or ADE of Streptomycetes origin has been purified or characterized for substrate specificity, despite of the fact that these organisms are the major producers of nucleoside antibiotics as
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Fig. 4. Protein sequence alignment of adenosine deaminases and adenine deaminases. The alignment was created using ESPript [23]. A box indicates an adenyl-deaminase novel motif. The nine amino acids important for ADA activity (numbered according to murine ADA) are His15, His17, Asp19, Gly184, His214, Glu217, His238, Asp295, and Asp296 as indicated by (⁄). The amino acids that distinguish ADA from ADE are indicated by black circles (d). For simplicity, only the catalytically important residues and residues that distinguish ADA from ADE as discussed in the text are highlighted. Absolutely conserved residues are shown in black and residues conserved in at least three of the sequence are shown in gray. Secondary structural elements corresponding to murine ADA (1A4L) are presented on the top of the alignment. Murine ADA (PO3958); Human ADA (POO813); E. coli ADA (Q8X661); Mycobacterium tuberculosis ADA (A5U7Y8); S. coelicolor ADA (CAB66224); A. nidulans ADE (AF123460 or AAL56636); S. pombe ADE (CAB91177); S. cerevisiae ADE (NP_014258 or CAA96024).
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secondary metabolites. For instance, two of the most potent inhibitors of ADA, coformycin and 20 -deoxycoformycin, are isolated from Streptomyces antibioticus [17]. Since nucleoside antibiotics often inhibit enzymes involved in nucleoside/nucleotide, RNA and DNA metabolism, antibiotic resistance has been shown to be a pre-requisite for antibiotic production. Since no ADA or ADE of Streptomycetes origin has been biochemically characterized, it is unclear whether enzymes in the salvage pathway from this organism may have evolved differently by altering their substrate/inhibitor specificity to avoid the inhibitory effect of antibiotics. ADA, ADE and AMP deaminases are closely related enzymes that utilize a similar catalytic machinery to catalyze mechanistically related reactions. Phylogenic analysis of amino acid sequences of various gene products suggests that these proteins belong to the adenyl-deaminase family [6]. This family contains five subfamilies, which include ADA, ADE, AMP deaminase, adenosine deaminase-like (ADAL), and adenosine deaminase-related growth factors (ADGF) subfamilies. Members of this family share a novel motif consisting of methionine (or iso/leucine), proline, lysine and glycine (MPKG), although these residues are not part of the catalytic machinery (Fig. 4). ADGF proteins contain all four of these residues, while ADAL and ADE proteins contain only the first three. In most ADA, only PK residues are conserved. In addition to this novel motif, eight of the catalytically active residues are highly conserved in all five protein subfamilies. S. coelicolor adenosine deaminase and adenine deaminase are divergently related enzymes By analyzing amino acid sequences of three characterized ADAs and three ascomycete ADEs, Ribard et al. [9] has suggested that some rules exist which can be used to differentiate between ADA and ADE. Asp19, Ser103, Ala183 and Gly184 (numbered according to the murine ADA) are characteristic of ADA, whereas Glu, Asp, Asp or Ser and Ser, respectively, are characteristic of ADE (Table 3). Of these four residues, only two residues, namely Asp and Gly in ADA or Glu and Ser in ADE, are part of the catalytic machinery (Fig. 4). Since SCO4901 contains Asp, Ala (rather than Ser as in ADA or Asp as in ADE), Ala and Gly, it is similar, but not identical, to the sequence of ADA than to that of ADE (Table 3). The purified recombinant enzyme deaminates adenosine and 20 -deoxyadenosine but not AMP. Although adenine is also deaminated by the enzyme, its rate of deamination occurs 103 slower than that of adenosine (Table 2). These data strongly suggest that the purified ScADA enzyme is an adenosine deaminase. The empirical rule suggested by Ribard et al. [9] appears to work in predicting whether a protein is ADA or ADE as reported in this study; how-
Table 3 Conserved residues that distinguish adenosine deaminase from adenine deaminase. ADA
ADE
SCO4901
Asp19 Ser103 Ala183 Gly184
Glu Asp Asp or Ser Ser
Asp Ala Ala Gly
The table is based on protein alignment of the murine, human and E. coli ADAs and A. nidulans, S. pombe and S. cerevisiae ADEs by Ribard et al. [9].
Table 4 Determination of the zinc content of S. coelicolor adenosine deaminase.
ScADA
Protein subunit (nmoles)
Zinc (nmoles)
Ratio Zinc/Protein subunit
1.7
1.2
0.7/1
ever, the structural differences between ADA and ADE are likely to be more subtle. ADA and ADE proteins show overall sequence similarity (approximately 25% identity and 40% similarity), adopt similar secondary structure as predicted by protein secondary structure prediction method, share similar catalytic machinery and probably form a similar (a/b)8 barrel with five additional helices. For some ADAs that have been characterized, such as A. oryzae and calf intestinal ADAs, both adenosine and adenine are substrates of the enzymes [16]. Both bind to the enzyme with similar value of Km, although adenine is deaminated 103–104 slower than that of adenosine. Thus, it is likely that ADA and ADE proteins are divergently related enzymes that have evolved from a common protein scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. If this is the case, it should be possible to change the substrate specificity of ADA to prefer adenine and thereby convert ADA to ADE or vice versa by in vitro or in vivo mutagenesis. Using a common fold in enzymes, the a/b barrel, as the scaffold, Altamirano et al. [18] were able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol phosphate synthase by in vitro evolution. In addition, protein alignment by Ribard et al. [9] also shows that two distinct families of adenine deaminases exist in nature. ADEs from biochemically characterized B. subtilis and E. coli bear no sequence similarity to the three ascomycete ADEs or ADAs and thus are in a branch of their own [9]. Furthermore, B. subtilis and E. coli ADEs are highly specific for adenine; neither adenosine nor other nucleosides are substrates for these enzymes [19–20]. Thus, it appears that the deaminase activity of ADEs from B. subtilis and E. coli and ADEs from ascomycetes may have evolved independently by convergent evolution. Proposed catalytic mechanism Protein alignment of ADAs from ten different organisms by Maier et al. [6] reveals that all of the active site residues are conserved. Of these nine highly conserved residues, three are involved in metal coordination, five interact with the purine ring and one interacts with the ribosyl moiety of adenosine. Eight of these nine conserved active site residues are also conserved in ScADA. The only non-conserved residue in ScADA is Asn317, which replaced Asp296 in the murine ADA (Figs. 4 and 5). Since ScADA was found to require zinc for activity, the three conserved histidines in ScADA, namely His35, His37 and His224, are likely to be involved in coordination of the zinc atom at the active site as observed in other ADAs [3,15]. The remaining six residues are involved substrate binding and/or catalysis. The catalytic mechanism of the ScADA enzyme is proposed to occur by a similar addition–elimination mechanism as proposed for murine ADA and S. cerevisiae AMP deaminase [3,21] (Fig. 5). The tetrahedral intermediate is formed by the direct attack of a water molecule to the C6 of adenosine. Glu227 further facilitates the formation of a tetrahedral C6 by donating a hydrogen bond to N1 of the purine ring and reduces the double bond character of the N1–C6 double bond. Gly197 and Asn317 of ScADA interact with the substrate, tetrahedral intermediate and product in the ES, EI and EP complexes by forming hydrogen bonds with N-3 and N-7, of the purine ring, respectively. The structure of the transition state formed at the active site of ScADA should be significantly distinct from that of murine ADA in the degree of N1 protonation. Recently, Schramm and coworkers [22] have demonstrated that similar enzymatic reactions catalyzed by proteins with high sequence homology and 100% conservation at catalytic site residues can have significantly distinct transition states. Elimination of amino groups from the tetrahedral intermediate results in formation of inosine. Similar to ScADA, the putative ADA from Mycobacterium tuberculosis (A5U7Y8) also contains Asn at this po-
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Fig. 5. Mechanism of the reaction catalyzed by adenosine deaminases from S. coelicolor. The nine highly conserved catalytically active residues are shown. In ScADA, Asn317 replaced the Asp296 in murine ADA, which is also shown in this diagram in parenthesis. The addition–elimination mechanism assumes the formation of a tetrahedral intermediate in the EI complex.
sition and probably is an adenosine deaminase (Fig. 4). The last remaining conserved residue, Asp19, is proposed to be involved in the formation hydrogen bonds with the 30 and 50 hydroxyl groups of the ribosyl moiety of adenosine. Conclusions In conclusion, this work describes the recombinant expression, purification and characterization of a putative ADA from S. coelicolor. The enzyme has been purified to homogeneity. The molecular weight of the native enzyme is 182 KDa and consists of four identical subunits, each with a molecular weight of 48.4 KDa. Adenosine and 20 -deoxyadenosine are substrates for the enzyme. Since adenine is deaminated 103 slower by the enzyme, when compared to that of adenosine, these data strongly show that the purified enzyme is an ADA and not an ADE. Similar to all other ADAs, ScADA is inhibited by 20 -deoxycoformycin. For S. antibioticus that produces coformycin and 20 -deoxycoformycin as nucleoside antibiotics, this organism is likely to possess a novel ADA that is resistant to inhibition by coformycin and 20 -deoxycoformycin. Acknowledgments This work was supported in part by the Thailand Research Fund, the Office of the Higher Education Commission (contact number MRG4680105) and the Kasetsart University Research and Development Institute to S.P. We are grateful to Dr. Vilai Santisopasri, Dr. Nonlawat Boonyalai, Attawan Sunantakarnkij and Sinothai Poen for suggestions and technical assistance. We thank Nancy Reichenbach for critical reading of this manuscript. References [1] G. Cristalli, S. Costanzi, C. Lambertucci, G. Lupidi, S. Vittori, R. Volpini, E. Camaioni, Adenosine deaminase: functional implications and different classes of inhibitors, Med. Res. Rev. 21 (2001) 105–128. [2] H. Kaneijam, T. Tanaka, Y. Nojima, S.F. Schlossman, C. Morioto, Direct association of adenosine deaminase with a T cell activation antigen, CD26, Science 261 (1993) 466–469. [3] D.K. Wilson, F.B. Rudolph, F.A. Quiocho, Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutation, Science 252 (1991) 1278–1284. [4] B. Even, R. Wofenden, A potential transition state analog for adenosine Deaminase, J. Am. Chem. Soc. 92 (1970) 4751–4752. [5] A.G. Chapman, D.E. Atkinson, Stabilization of adenylate energy charge by the adenylate deaminase reaction, J. Biol. Chem. 248 (1973) 8309–8312.
[6] S.A. Maier, J.R. Galellis, H.E. Mcdermid, Phylogenetic analysis reveals a novel protein family closely related to adenosine deaminase, J. Mol. Evol. 61 (2005) 776–794. [7] T. Weber, K. Welzel, S. Pelzer, A. Vente, W. Wohlleben, Exploiting the Genetic potential of polyketide producing Streptomycetes, J. Biotechnol. 106 (2003) 221–232. [8] S.D. Bentley, K.F. Chater, A.M. Gerdeno-Tarraga, G.L. Challis, N.R. Thomson, C.W. James, D.E. Harris, M.A. Quail, H. Keiser, D. Harper, A. Bateman, S. Brown, G. Chandra, C.W. Chen, M. Collins, A. Cronin, A. Fraser, A. Goble, J. Hidalgo, T. Hornsby, S. Howarth, C.H. Huang, T. Kieser, L. Larke, L. Murphy, K. Oliver, S. O’Neil, E. Rabbinowitsch, M.A. Rajandream, K. Rutherford, S. Rutter, K. Seeger, D. Saunders, S. Sharp, R. Squares, S. Squares, K. Taylor, T. Warren, A. Wietzorrek, J. Woodward, B.G. Barrell, J. Parkhill, D.A. Hopwood, Complete genome sequence of the model actinomyces Streptomyces coelicolor A3, Nature 417 (2002) 141–147. [9] C. Ribard, M. Rochet, B. Labedan, B. Daignan-Fornier, P. Alzari, C. Scazzochio, N. Oestreicher, Sub-families of a/b barrel enzymes: a new adenine deaminase family, J. Mol. Biol. 334 (2003) 1117–1131. [10] T. Kieser, M.J. Bibb, M.J. Buttner, K.F. Chater, D.A. Hopwood, Practical Streptomyces Genetics, John Innes Foundation, Norwich, England, 2000. [11] P. Howhan, S. Pornbanlualap, Cloning and effective induction of Escherichia coli nucleoside diphosphate kinase by lactose, SciAsia 29 (2003) 347–353. [12] J. Cory, R.J. Suhadolnik, Structural requirements of nucleosides for binding by adenosine deaminase, Biochemistry 4 (1965) 1729–1732. [13] D.E. Ingolia, C.Y. Yeung, I.F. Orengo, M.L. Harrison, E.G. Frayne, F.B. Rudolph, R.E. Kellems, Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line, J. Boil. Chem. 260 (1985) 13261– 13267. [14] W.P. Schrader, A.R. Stacy, Immunoassay of the adenosine deaminase complexing proteins of human tissues and body fluids, J. Biol. Chem. 254 (1979) 11958–11963. [15] E.T. Larson, W. Deng, B.E. Krumm, A. Napuli, N. Mueller, W.C. Van Voorthis, F.S. Buckner, E. Fan, A. Lauricella, G. DeTitta, J. Luft, F. Zucker, W.G. Hol, C.L. Verlinde, E.A. Merritt, Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity, J. Mol. Biol. 381 (2008) 975–988. [16] R. Wolfenden, J. Kaufman, J. B Macon, Ring-modified substrates of adenosine deaminase, Biochemistry 8 (1969) 2412–2415. [17] R.J. Suhadolnik, Nucleosides as Biological Probes, John Wiley & Sons, Interscience, New York, 1979. [18] M.M. Altamirano, J.M. Blackburn, C. Aguayo, A. Fersht, Directed evolution of a-barrel scaffold, Nature 404 (2000) 617– new catalytic activity using the b 622. [19] L.A. Heppel, J. Hurwitz, B.L. Horeckek, Adenine deaminase of Azotobacter vinelandii, J. Am. Chem. Soc. 79 (1957) 630–633. [20] H. Matsui, M. Shimaoka, H. Kawasaki, Y. Takenaka, O. Kurahashi, Adenine deaminase activity of the yic P gene product of Escherichia coli, Biosci. Biotechnol. Biochem. 65 (2001) 1112–1118. [21] D.J. Merkler, P.C. Kline, P. Weiss, V.L. Schramm, Transition-state analysis of AMP deaminase, Biochemistry 32 (1993) 12993–13001. [22] M. Luo, V. Sigh, E.A. Taylor, V.L. Schramm, Transition-state variation in human, bovine and Plasmodium falciparum adenosine deaminase, J. Am. Chem. Soc. 129 (2007) 8008–8017. [23] P. Gouet, X. Robert, E. Courcelle, ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins, Nucleic Acids Res. 31 (2003) 133320–133323.
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Adenosine deaminase from Streptomyces coelicolor: Recombinant expression, purification and characterization Somchai Pornbanlualap ⇑, Pornchanok Chalopagorn Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand
a r t i c l e
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Article history: Received 11 January 2011 and in revised form 28 March 2011 Available online 12 April 2011 Keywords: Streptomyces coelicolor Adenosine deaminase Adenine deaminase Transition state 20 -Deoxycoformycin Divergent evolution
a b s t r a c t The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr = 48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s1at 30 °C. Since adenine is deaminated 103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-b-D-arabinofuranosyl-adenine (ara-A), 50 -AMP, 50 -ADP and 50 -ATP, are not substrates for the enzyme. Coformycin and 20 deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common a/b barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. Ó 2011 Elsevier Inc. All rights reserved.
Introduction Adenosine deaminase (ADA, EC 3.5.4.4) catalyzes the irreversible deamination of adenosine to inosine and ammonia. This enzyme has been found in a wide variety of microorganisms, plants, invertebrates and mammals. It is an indispensable enzyme in purine metabolism. In humans, this enzyme is required for B and T-cell development and plays a central role in the maintenance of a competent immune system [1]. Genetic deficiency of ADA results in a disease known as severe combined immunodeficiency disease (SCID), which is characterized by a lack of T- and B-lymphocytes. Children with this disease have no detectable lymphocytes and are subject to recurrent infections [2]. The crystal structure of murine ADA shows that this protein is a zinc metalloenzyme that displays a typical ab-barrel fold, with eight central b-strands and eight peripheral a-helices [3]. In addition to this central motif, the structure contains five additional a-helices. The catalytic mechanism of deamination has been ⇑ Corresponding author. Fax: +66 2 561 4627. E-mail address: [email protected] (S. Pornbanlualap). 1046-5928/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2011.04.003
suggested to proceed by the direct water attack on the C-6 position of the purine ring, resulting in the formation of a tetrahedral intermediate [4] (Fig. 1). Thus, 6-hydroxyl-1,6-dihydropurine riboside (HDPR) and the naturally occurring nucleoside antibiotic, 20 -deoxycoformycin, whose structures mimic the hypothetical tetrahedral transition state intermediate, bind tightly to the enzyme, with Ki of 1013 and 1012 M, respectively. Adenine deaminase (ADE) and AMP deaminase are enzymes mechanistically related to ADA. ADE is also involved in the purine salvage pathway by deamination of adenine to hypoxanthine and ammonia. AMP deaminase catalyzes the formation of IMP and ammonia from AMP and is involved in regulating adenine nucleotide levels in eukaryotes [5]. Recent phylogenic analysis of amino acid sequences of various gene products suggests that ADAs, ADEs, AMP deaminases, adenosine deaminase-like (ADAL), and adenosine deaminase-related growth factors (ADGF) are a group of proteins that belong to the adenyl-deaminase family [6]. Members of this family share a novel motif consisting of methionine (or iso/leucine), proline, lysine and glycine (MPKG). In addition to this novel motif, the catalytically active residues are highly conserved in all five protein subfamilies.
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Fig. 1. (A) Reaction coordinates for deamination of adenosine to inosine catalyzed by adenosine deaminase. The reaction occurs by direct water attack on C6 of adenosine to generate a tetrahedral intermediate. The relative energy along the reaction coordinates is arbitrary with the highest energetic point as the transition state. As substrate is transformed into the transition state and changes in the [C6–Ohydroxyl], [C6–N6], [C1–N1] and [N1–H] bond lengths occurs. Modified from Luo et al. [22]. (B) Structure of competitive inhibitor, 6R-hydroxyl-1,6-dihydropurine riboside (HDPR), formed by covalent hydration of purine riboside. (C) Structure of 8-(R)-coformycin, a nucleoside antibiotic and a transition state inhibitor. The sugar substituent R in coformycin is ribose and in 20 -deoxycformycin is 20 -deoxyribose.
Plasmid construction
Streptomycetes are a group of filamentous Gram-positive, soilinhabiting bacteria which have captured enormous screening interest because of their ability to produce and secrete a variety of antibiotics and extracellular proteins [7]. Among the diverse antibiotics produced by Streptomycetes are the nucleoside antibiotics. The sequence of the genome of Streptomyces coelicolor A3(2) has been predicted to contain genes encoding more than 7500 proteins, many of which have yet to be studied and defined [8]. SCO4901 is one of these genes that had been annotated in data bases as a ‘‘putative’’ adenosine deaminase. However, since ADA and ADE proteins have been shown to have approximately the same size, exhibit overall sequence similarity, contain similar folding topology and share the same catalytic machinery, it is often difficult to unambiguously distinguish one from the other. Characterization of the ADE sequences from Aspergillus nidulans, Saccharomyces cerevisiae and Schilzosaccharomyces pombe shows that these proteins are more closely related to the biochemically characterized ADAs rather than Bacillus subtilis and Escherichia coli ADEs. Based on sequence alignment of ascomycete ADEs and the characterized ADAs, a structural rule has been proposed to distinguish whether a protein is an ADA or an ADE [9]. Asp19, Ser103, Ala183, and Gly184 (numbered according to murine ADA) have been proposed to be characteristic of ADA, whereas Glu, Asp, Asp or Ser, and Ser at the same position are characteristic of ADE. Although Streptomycetes are the major producers of nucleoside antibiotics and many of nucleoside antibiotics exhibit inhibitory effects on purine salvaging enzymes, few enzymes of Streptomycetes origin have been biochemically characterized. Therefore, this report describes the expression, characterization of substrate specificity and inhibition of adenosine deaminase from S. coelicolor (ScADA) by transition state analogs.
Genomic DNA was purified from S. coelicolor cells grown in yeast extract-malt extract (YEME) containing 34% sucrose, 1% glucose and 0.5% MgCl2 at 30 °C as described by Hopwood et al. [10]. The concentration of genomic DNA was measured in a UV spectrophotometer (at 260 nm). The size of genomic DNA was analyzed on 1% agarose gels. The forward primer (ADA-1: 50 -TTTAAGCTTCATATG ACGAGCCGAAGCACCGAG-30 , where the underline indicates the added HindIII and NdeI linkers) and the reverse primer (ADA2: 50 -TTTAGATCTTCAGCCGTCG GATTCCGAAGA-30 , where the underline indicates the added BglII linker) were designed according to the putative adenosine deaminase gene sequence from S. coelicolor (Genebank Accession No. SCO4901). The complete coding region of the gene was amplified by PCR with ADA-1 and ADA-2 primers, using chromosomal DNA prepared from S. coelicolor as the template and Taq DNA polymerase. PCR was performed as follows: initial denaturing at 93 °C for 5 min; 30 cycles of denaturation at 93 °C for 1 min, annealing at 68 °C for 1 min, and additional extension at 72 °C for 1.5 min, and additional extension at 72 °C for 10 min. The PCR product was gel purified with a PCR purification kit (Qiagen) and ligated into the pGEM-T vector (Promega). After transformation into competent E. coli JM109, recombinant plasmid, pGEM-ada, was purified from positive clones by the alkali method and sequenced to confirm that no mutations were introduced by the PCR amplification step. After a double digestion of pGEM-ada with NdeI and BglII, the 1.2 kb fragment was gel purified and ligated into pET-15b, which had been previously double digested with NdeI and BamHI. The ligation mixture was transformed into E. coli BL21 (DE3) and selected on LB containing 60 lg/ml ampicillin.
Materials and methods
Protein purification
Materials
For purification of adenosine deaminase, 5 ml of E. coli BL21 (DE3) carrying pET-ada from overnight grown culture in LB supplemented with 60 lg/ml ampicillin was transferred to one liter of LB plus ampicillin. The culture was allowed to grow to an OD600 nm of 0.5 and induced by addition of lactose at a final concentration of 1 mM, since lactose had been shown to be as an effective inducer as IPTG [11]. After five additional hours of growth, cells were harvested by centrifugation at 10,000g at 4 °C, suspended in lysis buffer [20 mM Tris–HCl (pH 8.0), 50 mM KCl, 1 mM EDTA, 0.5% Tween
Adenosine, 20 -deoxyadenosine, adenine, 9-b-D-arabinofuranosyladenine (ara-A), and IPTG were from Sigma/Aldrich. 20 -Deoxycoformycin and coformycin were provided by Dr. Robert J. Suhadolnik, Temple University School of Medicine (USA). Nickelagarose was from Qiagen. S. coelicolor A3(2) was a generous gift from Dr. Keith Chater, John Innes Institute (UK). E. coli strain BL21 (DE3) and pET-15b were from Novagen.
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20 and 1 mM PMSF] and disrupted by sonication. The sonicator was programmed to provide 15 s pulses with a 10 s pause for a total period of 30 min. After centrifugation at 12,000g for 30 min, the enzyme in the supernatant was precipitated by addition of ammonium sulfate at 80% saturation. Precipitated proteins were collected by centrifugation at 10,000g, dissolved in 4 ml of lysis buffer and dialyzed four times against 3 l of buffer containing 50 mM potassium phosphate (pH 7.5). The dialyzed protein was added onto Ni2+-NTA resin (1 ml bed volume), which had been previously equilibrated with buffer B [20 mM Tris–HCl (pH 8), 500 mM KCl, and 0.1% Triton X-100]. After washing the column with 4 ml of buffer B, the polyhistidine-tagged enzyme was eluted by washing with buffer B containing 20, 100 and 250 mM imidazole, respectively. The enzyme eluted from column was analyzed on 10% SDS–PAGE. Assay for adenosine deaminase activity The activity of adenosine deaminase was assayed spectrophotometrically by measuring the decrease in absorbance at 265 nm on conversion of adenosine to inosine with a Shimazu spectrophotometer. One milliliter reactions containing 0.04–0.08 lmole of adenosine in 50 mM potassium phosphate were incubated for 1 min in a cuvette prior to the addition of enzyme. The amount of product formed was calculated using a molar extinction coefficient of 8.4 mM1 [12]. The same wavelength and molar extinction coefficients were used for determination of the Km and Vmax for ara-A, 20 -deoxyadenosine, adenine, AMP, ADP, and ATP. One unit of enzyme activity is defined as the amount of enzyme needed to convert 1 lmole of substrate to product at 37 °C in one minute. Protein concentration was determined by the Bradford method, using bovine serum albumin as standard. Analysis of kinetic data Kinetic data were fitted to the appropriate equations using the Sigma Plot program. The initial velocity (v) obtained by varying the concentration of substrate was fitted to the equation: v = Vmax [S]/Km + [S], where Vmax is the maximum velocity, [S] is substrate concentration, and Km is the Michaelis constant. Kinetic data obtained from competitive inhibition were fitted to the equation: v = Vmax [S]/Km (1+[I]/Ki) + [S], where [I] is the concentration of inhibitor and Ki is the inhibition constant. Molecular weight estimation The elution position of active ADA relative to protein of known molecular weight was determined by size exclusion chromatography, using HiPrep 16/60 Sephacryl S-200HR column (Pharmacia). A solution (0.5 ml) containing 0.4 mg/ml of ADA, 10 units of pyruvate kinase (237 kDa), 3 mg/ml conalbumin (75 kDa), 4 mg/ml ovalbumin (43 kDa), 50 mM KH2PO4 (pH 7.0) and 0.15 M NaCl was applied to a column which had been equilibrated with the same buffer but lacking the enzymes. The flow rate was 1 ml/minute and the elution profiles were monitored at 280 nm by UV absorption. The elution position of ADA was detected by the deamination assay described above. Metal analysis Purified protein samples were analyzed for zinc content with Varian atomic absorption spectrophotometer model AA280 FS at 213.8 nm. Protein was quantified using the predicted molar extinction coefficient (at 44,265 M1 cm1 at 280 nm) and by the Bradford method, using bovine serum albumin as standard.
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Results Cloning of the putative adenosine deaminase gene Using the ADA-1 and ADA-2 primers, PCR reactions resulted in a single band of the correct length 1.2 kb. The nucleotide sequence of gel-purified DNA was confirmed as a putative adenosine deaminase from S. coelicolor by sequencing before it was cloned into pET-15b. The resultant plasmid, pET-ada, was expected to express a protein of 396 amino acids with 20 additional amino acids, including a polyhistidine tag at the N-terminus and a predicted molecular mass of 45,987 Da. The crystal structure of murine ADA (MuADA) shows that the enzyme folds as an eight stranded parallel ab barrel [3]. Since the active site of proteins with (a/b)8 barrel domain is located at the COOH-terminal face of b barrel, addition of polyhistidine at the NH2-terminus of enzyme was expected to have little or no effect on the catalytic activity. Purification of the adenosine deaminase A major band with molecular weight of approximately 48 KDa was observed after five hours of induction with 1 mM lactose (Fig. 2A). The purification of recombinant S. coelicolor adenosine deaminase (ScADA) from one liter of E. coli BL21(DE3) carrying the pET-ada cell culture resulted in a total of 16.5 mg protein with the specific activity of 2.07 lmole/min/mg (Table 1) and 85% homogeneity as estimated from denaturing polyacrylamide gel electrophoresis (Fig. 2A). When the molecular weight of the enzyme was estimated by gel-exclusion chromatography using proteins of known masses as standards, ScADA eluted at an apparent molecular weight of 182,000 (Fig. 2B). These values are consistent with the active enzyme being composed of four subunits with identical molecular weight. This is in contrast to murine, human and plasmodial ADAs, where the enzymes are active as monomers [13–15]. Substrate/inhibitor specificity and zinc requirement The kinetic behavior of the recombinant enzyme with adenosine and 20 -deoxyadenosine may be reasonably well described by the simple Michaelis–Menten model. The Km, Ki, and Vmax values of purified enzyme for substrates/inhibitors are shown in Table 2. Substrate specificity studies show that adenosine is the best substrate with Vmax of 15.4 (lmole/min/mg) and turnover number (kcat) of 11.5 s1. Substitution of the hydroxyl group at the 20 -position with hydrogen (i.e., 20 -deoxyadenosine) is moderately tolerated, resulting in a decrease of relative Vmax to 17.5% as compared to adenosine and more than a 15-fold increase in Km. Removal of the ribosyl moiety from adenosine (i.e., adenine) or inversion of the hydroxyl group at the 20 -position (i.e., ara-A) results in almost complete loss of activity as substrate. (Vmax < 0.06%). Addition of the phosphate group to the 50 -position of the ribosyl moiety (i.e., 50 -AMP, 50 -ADP, and 50 -ATP) also results in complete loss of activity as substrate. Since it is not deaminated by the enzyme, adenine was tested as an inhibitor of the enzyme. The result shows that adenine is a poor inhibitor of the enzyme with a Ki > 1 mM. Aspergillus oryzae and calf intestinal ADAs bind to adenosine and adenine with similar Km, although the former is deaminated 103–104 faster than the latter [16]. Coformycin and 20 -deoxycoformycin, whose structures mimic the transition state of the deamination reaction, are potent competitive inhibitors of the enzyme, with Ki of 2.5 1010 and 3.4 109 M, respectively (Fig. 3). Substrate specificity and inhibitor studies showed that the presence of the hydroxyl group at the C-20 position of the ribosyl moiety appears to contribute to tight
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Fig. 2. Analysis of the purified adenosine deaminase by polyacrylamide gel electrophoresis and gel exclusion chromatography. Panel A: lane 1, standard protein markers; lane 2, cell-free extract before induction; lane 3, cell-free extract after induced with lactose; lane 4, 5 lg of protein isolated following Ni–NTA chromatography step. Panel B: Elution position of native adenosine deaminase relative to proteins of known molecular weight (see Materials and methods section).
Table 1 Summary of the purification of S. coelicolor adenosine deaminase. Step
Total protein (mg)
Total unit (U)
Specific activity (l mole/min/mg)
Fold purification
Recovery (%)
Cell-free extract 0–80% (NH4)2SO4 Ni2+-NTA affinity
120 38 16.5
84.8 36.5 34.2
0.35 0.96 2.07
1.0 2.7 5.9
100 43 40
Table 2 Comparison of Km, Ki, and Vmax values for the interaction of adenosine analogs with adenosine deaminase from S. coelicolor. Substrate or inhibitor Substrates Adenosine 20 -Deoxyadenosine Ara-A Adenine 50 -AMP 50 -ADP 50 -ATP Inhibitors Coformycin 20 -Deoxycoformycin Adenine
Km (lM)
Ki (lM)
29 560
Vmax (l mole/min/mg)
Vmax (%)
15.4 2.7 <0.011 <0.011 ND ND ND
100.0 17.5 <0.06 <0.06
0.00025 0.0034 >1000
ND – not detectable. 1 The deamination was too slow to allow determination of Km.
binding of the substrate or inhibitor to the enzyme, since ribonucleosides (adenosine and coformycin) bind tighter to the enzyme than their deoxyribonucleoside counterparts (20 -deoxyadenosine and 20 -deoxycoformycin). In contrast, ADA isolated from other sources has been reported to bind slightly tighter to 20 -deoxycoformycin than coformycin. No slow onset of inhibition of ScADA by coformycin or 20 -deoxycoformycin was observed under our experimental conditions. The purified ScADA requires zinc for catalytic activity. In the absence of 1,10-phenanthroline, ScADA retained virtually 100% of activity for 60 min (data not shown). When the enzyme was incubated 0.7 mM of 1,10-phenanthroline, the enzyme lost approximately 90% of its catalytic activity after 60 min of incubation. Analysis of the metal content of purified enzyme by atomic absorption spectroscopy shows that the enzyme contains approximately 0.7 mole of tightly bound zinc/mole of subunits (Table 4). The less than the expected 1:1 ratio of zinc/ subunit of enzyme is perhaps due to insufficient amount of zinc presence in the growth medium needed to be incorporated into the expressed enzyme upon induction with lactose.
Fig. 3. The effect of 20 -deoxycoformycin (20 -dCF) on the initial rate of product formation by adenosine deaminase. Inhibition of ADA by 20 -dCF was measured in 50 mM potassium phosphate buffer, pH 7.0. The initial rates were measured by the decrease in optical density at 265 nm. The data points were fitted to an equation for competitive inhibition.
Discussion The purposes of this study were to (i) determine whether SCO4901 encodes for ADA or ADE, (ii) purify the recombinant protein and determine its subunit structure, (iii) characterize substrate and inhibitor specificity of the enzyme and (iv) elucidate the evolutionally relationship between ADA and ADE. To this date, no ADA or ADE of Streptomycetes origin has been purified or characterized for substrate specificity, despite of the fact that these organisms are the major producers of nucleoside antibiotics as
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171
Fig. 4. Protein sequence alignment of adenosine deaminases and adenine deaminases. The alignment was created using ESPript [23]. A box indicates an adenyl-deaminase novel motif. The nine amino acids important for ADA activity (numbered according to murine ADA) are His15, His17, Asp19, Gly184, His214, Glu217, His238, Asp295, and Asp296 as indicated by (⁄). The amino acids that distinguish ADA from ADE are indicated by black circles (d). For simplicity, only the catalytically important residues and residues that distinguish ADA from ADE as discussed in the text are highlighted. Absolutely conserved residues are shown in black and residues conserved in at least three of the sequence are shown in gray. Secondary structural elements corresponding to murine ADA (1A4L) are presented on the top of the alignment. Murine ADA (PO3958); Human ADA (POO813); E. coli ADA (Q8X661); Mycobacterium tuberculosis ADA (A5U7Y8); S. coelicolor ADA (CAB66224); A. nidulans ADE (AF123460 or AAL56636); S. pombe ADE (CAB91177); S. cerevisiae ADE (NP_014258 or CAA96024).
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secondary metabolites. For instance, two of the most potent inhibitors of ADA, coformycin and 20 -deoxycoformycin, are isolated from Streptomyces antibioticus [17]. Since nucleoside antibiotics often inhibit enzymes involved in nucleoside/nucleotide, RNA and DNA metabolism, antibiotic resistance has been shown to be a pre-requisite for antibiotic production. Since no ADA or ADE of Streptomycetes origin has been biochemically characterized, it is unclear whether enzymes in the salvage pathway from this organism may have evolved differently by altering their substrate/inhibitor specificity to avoid the inhibitory effect of antibiotics. ADA, ADE and AMP deaminases are closely related enzymes that utilize a similar catalytic machinery to catalyze mechanistically related reactions. Phylogenic analysis of amino acid sequences of various gene products suggests that these proteins belong to the adenyl-deaminase family [6]. This family contains five subfamilies, which include ADA, ADE, AMP deaminase, adenosine deaminase-like (ADAL), and adenosine deaminase-related growth factors (ADGF) subfamilies. Members of this family share a novel motif consisting of methionine (or iso/leucine), proline, lysine and glycine (MPKG), although these residues are not part of the catalytic machinery (Fig. 4). ADGF proteins contain all four of these residues, while ADAL and ADE proteins contain only the first three. In most ADA, only PK residues are conserved. In addition to this novel motif, eight of the catalytically active residues are highly conserved in all five protein subfamilies. S. coelicolor adenosine deaminase and adenine deaminase are divergently related enzymes By analyzing amino acid sequences of three characterized ADAs and three ascomycete ADEs, Ribard et al. [9] has suggested that some rules exist which can be used to differentiate between ADA and ADE. Asp19, Ser103, Ala183 and Gly184 (numbered according to the murine ADA) are characteristic of ADA, whereas Glu, Asp, Asp or Ser and Ser, respectively, are characteristic of ADE (Table 3). Of these four residues, only two residues, namely Asp and Gly in ADA or Glu and Ser in ADE, are part of the catalytic machinery (Fig. 4). Since SCO4901 contains Asp, Ala (rather than Ser as in ADA or Asp as in ADE), Ala and Gly, it is similar, but not identical, to the sequence of ADA than to that of ADE (Table 3). The purified recombinant enzyme deaminates adenosine and 20 -deoxyadenosine but not AMP. Although adenine is also deaminated by the enzyme, its rate of deamination occurs 103 slower than that of adenosine (Table 2). These data strongly suggest that the purified ScADA enzyme is an adenosine deaminase. The empirical rule suggested by Ribard et al. [9] appears to work in predicting whether a protein is ADA or ADE as reported in this study; how-
Table 3 Conserved residues that distinguish adenosine deaminase from adenine deaminase. ADA
ADE
SCO4901
Asp19 Ser103 Ala183 Gly184
Glu Asp Asp or Ser Ser
Asp Ala Ala Gly
The table is based on protein alignment of the murine, human and E. coli ADAs and A. nidulans, S. pombe and S. cerevisiae ADEs by Ribard et al. [9].
Table 4 Determination of the zinc content of S. coelicolor adenosine deaminase.
ScADA
Protein subunit (nmoles)
Zinc (nmoles)
Ratio Zinc/Protein subunit
1.7
1.2
0.7/1
ever, the structural differences between ADA and ADE are likely to be more subtle. ADA and ADE proteins show overall sequence similarity (approximately 25% identity and 40% similarity), adopt similar secondary structure as predicted by protein secondary structure prediction method, share similar catalytic machinery and probably form a similar (a/b)8 barrel with five additional helices. For some ADAs that have been characterized, such as A. oryzae and calf intestinal ADAs, both adenosine and adenine are substrates of the enzymes [16]. Both bind to the enzyme with similar value of Km, although adenine is deaminated 103–104 slower than that of adenosine. Thus, it is likely that ADA and ADE proteins are divergently related enzymes that have evolved from a common protein scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. If this is the case, it should be possible to change the substrate specificity of ADA to prefer adenine and thereby convert ADA to ADE or vice versa by in vitro or in vivo mutagenesis. Using a common fold in enzymes, the a/b barrel, as the scaffold, Altamirano et al. [18] were able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol phosphate synthase by in vitro evolution. In addition, protein alignment by Ribard et al. [9] also shows that two distinct families of adenine deaminases exist in nature. ADEs from biochemically characterized B. subtilis and E. coli bear no sequence similarity to the three ascomycete ADEs or ADAs and thus are in a branch of their own [9]. Furthermore, B. subtilis and E. coli ADEs are highly specific for adenine; neither adenosine nor other nucleosides are substrates for these enzymes [19–20]. Thus, it appears that the deaminase activity of ADEs from B. subtilis and E. coli and ADEs from ascomycetes may have evolved independently by convergent evolution. Proposed catalytic mechanism Protein alignment of ADAs from ten different organisms by Maier et al. [6] reveals that all of the active site residues are conserved. Of these nine highly conserved residues, three are involved in metal coordination, five interact with the purine ring and one interacts with the ribosyl moiety of adenosine. Eight of these nine conserved active site residues are also conserved in ScADA. The only non-conserved residue in ScADA is Asn317, which replaced Asp296 in the murine ADA (Figs. 4 and 5). Since ScADA was found to require zinc for activity, the three conserved histidines in ScADA, namely His35, His37 and His224, are likely to be involved in coordination of the zinc atom at the active site as observed in other ADAs [3,15]. The remaining six residues are involved substrate binding and/or catalysis. The catalytic mechanism of the ScADA enzyme is proposed to occur by a similar addition–elimination mechanism as proposed for murine ADA and S. cerevisiae AMP deaminase [3,21] (Fig. 5). The tetrahedral intermediate is formed by the direct attack of a water molecule to the C6 of adenosine. Glu227 further facilitates the formation of a tetrahedral C6 by donating a hydrogen bond to N1 of the purine ring and reduces the double bond character of the N1–C6 double bond. Gly197 and Asn317 of ScADA interact with the substrate, tetrahedral intermediate and product in the ES, EI and EP complexes by forming hydrogen bonds with N-3 and N-7, of the purine ring, respectively. The structure of the transition state formed at the active site of ScADA should be significantly distinct from that of murine ADA in the degree of N1 protonation. Recently, Schramm and coworkers [22] have demonstrated that similar enzymatic reactions catalyzed by proteins with high sequence homology and 100% conservation at catalytic site residues can have significantly distinct transition states. Elimination of amino groups from the tetrahedral intermediate results in formation of inosine. Similar to ScADA, the putative ADA from Mycobacterium tuberculosis (A5U7Y8) also contains Asn at this po-
S. Pornbanlualap, P. Chalopagorn / Protein Expression and Purification 78 (2011) 167–173
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Fig. 5. Mechanism of the reaction catalyzed by adenosine deaminases from S. coelicolor. The nine highly conserved catalytically active residues are shown. In ScADA, Asn317 replaced the Asp296 in murine ADA, which is also shown in this diagram in parenthesis. The addition–elimination mechanism assumes the formation of a tetrahedral intermediate in the EI complex.
sition and probably is an adenosine deaminase (Fig. 4). The last remaining conserved residue, Asp19, is proposed to be involved in the formation hydrogen bonds with the 30 and 50 hydroxyl groups of the ribosyl moiety of adenosine. Conclusions In conclusion, this work describes the recombinant expression, purification and characterization of a putative ADA from S. coelicolor. The enzyme has been purified to homogeneity. The molecular weight of the native enzyme is 182 KDa and consists of four identical subunits, each with a molecular weight of 48.4 KDa. Adenosine and 20 -deoxyadenosine are substrates for the enzyme. Since adenine is deaminated 103 slower by the enzyme, when compared to that of adenosine, these data strongly show that the purified enzyme is an ADA and not an ADE. Similar to all other ADAs, ScADA is inhibited by 20 -deoxycoformycin. For S. antibioticus that produces coformycin and 20 -deoxycoformycin as nucleoside antibiotics, this organism is likely to possess a novel ADA that is resistant to inhibition by coformycin and 20 -deoxycoformycin. Acknowledgments This work was supported in part by the Thailand Research Fund, the Office of the Higher Education Commission (contact number MRG4680105) and the Kasetsart University Research and Development Institute to S.P. We are grateful to Dr. Vilai Santisopasri, Dr. Nonlawat Boonyalai, Attawan Sunantakarnkij and Sinothai Poen for suggestions and technical assistance. We thank Nancy Reichenbach for critical reading of this manuscript. References [1] G. Cristalli, S. Costanzi, C. Lambertucci, G. Lupidi, S. Vittori, R. Volpini, E. Camaioni, Adenosine deaminase: functional implications and different classes of inhibitors, Med. Res. Rev. 21 (2001) 105–128. [2] H. Kaneijam, T. Tanaka, Y. Nojima, S.F. Schlossman, C. Morioto, Direct association of adenosine deaminase with a T cell activation antigen, CD26, Science 261 (1993) 466–469. [3] D.K. Wilson, F.B. Rudolph, F.A. Quiocho, Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutation, Science 252 (1991) 1278–1284. [4] B. Even, R. Wofenden, A potential transition state analog for adenosine Deaminase, J. Am. Chem. Soc. 92 (1970) 4751–4752. [5] A.G. Chapman, D.E. Atkinson, Stabilization of adenylate energy charge by the adenylate deaminase reaction, J. Biol. Chem. 248 (1973) 8309–8312.
[6] S.A. Maier, J.R. Galellis, H.E. Mcdermid, Phylogenetic analysis reveals a novel protein family closely related to adenosine deaminase, J. Mol. Evol. 61 (2005) 776–794. [7] T. Weber, K. Welzel, S. Pelzer, A. Vente, W. Wohlleben, Exploiting the Genetic potential of polyketide producing Streptomycetes, J. Biotechnol. 106 (2003) 221–232. [8] S.D. Bentley, K.F. Chater, A.M. Gerdeno-Tarraga, G.L. Challis, N.R. Thomson, C.W. James, D.E. Harris, M.A. Quail, H. Keiser, D. Harper, A. Bateman, S. Brown, G. Chandra, C.W. Chen, M. Collins, A. Cronin, A. Fraser, A. Goble, J. Hidalgo, T. Hornsby, S. Howarth, C.H. Huang, T. Kieser, L. Larke, L. Murphy, K. Oliver, S. O’Neil, E. Rabbinowitsch, M.A. Rajandream, K. Rutherford, S. Rutter, K. Seeger, D. Saunders, S. Sharp, R. Squares, S. Squares, K. Taylor, T. Warren, A. Wietzorrek, J. Woodward, B.G. Barrell, J. Parkhill, D.A. Hopwood, Complete genome sequence of the model actinomyces Streptomyces coelicolor A3, Nature 417 (2002) 141–147. [9] C. Ribard, M. Rochet, B. Labedan, B. Daignan-Fornier, P. Alzari, C. Scazzochio, N. Oestreicher, Sub-families of a/b barrel enzymes: a new adenine deaminase family, J. Mol. Biol. 334 (2003) 1117–1131. [10] T. Kieser, M.J. Bibb, M.J. Buttner, K.F. Chater, D.A. Hopwood, Practical Streptomyces Genetics, John Innes Foundation, Norwich, England, 2000. [11] P. Howhan, S. Pornbanlualap, Cloning and effective induction of Escherichia coli nucleoside diphosphate kinase by lactose, SciAsia 29 (2003) 347–353. [12] J. Cory, R.J. Suhadolnik, Structural requirements of nucleosides for binding by adenosine deaminase, Biochemistry 4 (1965) 1729–1732. [13] D.E. Ingolia, C.Y. Yeung, I.F. Orengo, M.L. Harrison, E.G. Frayne, F.B. Rudolph, R.E. Kellems, Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line, J. Boil. Chem. 260 (1985) 13261– 13267. [14] W.P. Schrader, A.R. Stacy, Immunoassay of the adenosine deaminase complexing proteins of human tissues and body fluids, J. Biol. Chem. 254 (1979) 11958–11963. [15] E.T. Larson, W. Deng, B.E. Krumm, A. Napuli, N. Mueller, W.C. Van Voorthis, F.S. Buckner, E. Fan, A. Lauricella, G. DeTitta, J. Luft, F. Zucker, W.G. Hol, C.L. Verlinde, E.A. Merritt, Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity, J. Mol. Biol. 381 (2008) 975–988. [16] R. Wolfenden, J. Kaufman, J. B Macon, Ring-modified substrates of adenosine deaminase, Biochemistry 8 (1969) 2412–2415. [17] R.J. Suhadolnik, Nucleosides as Biological Probes, John Wiley & Sons, Interscience, New York, 1979. [18] M.M. Altamirano, J.M. Blackburn, C. Aguayo, A. Fersht, Directed evolution of a-barrel scaffold, Nature 404 (2000) 617– new catalytic activity using the b 622. [19] L.A. Heppel, J. Hurwitz, B.L. Horeckek, Adenine deaminase of Azotobacter vinelandii, J. Am. Chem. Soc. 79 (1957) 630–633. [20] H. Matsui, M. Shimaoka, H. Kawasaki, Y. Takenaka, O. Kurahashi, Adenine deaminase activity of the yic P gene product of Escherichia coli, Biosci. Biotechnol. Biochem. 65 (2001) 1112–1118. [21] D.J. Merkler, P.C. Kline, P. Weiss, V.L. Schramm, Transition-state analysis of AMP deaminase, Biochemistry 32 (1993) 12993–13001. [22] M. Luo, V. Sigh, E.A. Taylor, V.L. Schramm, Transition-state variation in human, bovine and Plasmodium falciparum adenosine deaminase, J. Am. Chem. Soc. 129 (2007) 8008–8017. [23] P. Gouet, X. Robert, E. Courcelle, ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins, Nucleic Acids Res. 31 (2003) 133320–133323.