Báo cáo khoa học: Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis pptx

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis Isaura Simo ˜ es 1,2 , Rosa ´ rio Faro 1 , Daniel Bur 3 , John Kay 4 and Carlos Faro 1,2 1 CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal 2 Biocant, Biotechnology Innovation Center, Cantanhede, Portugal 3 Actelion Pharmaceuticals Ltd, Allschwil, Switzerland 4 School of Biosciences, Cardiff University, UK Keywords aspartic proteinase; bacteria; pepsin-like Correspondence I. Simo˜ es, Biocant, Parque Tecnolo ´ gico de Cantanhede, Nu ´ cleo 4, Lote 3, 3060-197 Cantanhede, Portugal Fax: +351 231 419049 Tel: +351 231 419040 E-mail: isimoes@biocant.pt (Received 19 April 2011, revised 4 July 2011, accepted 8 July 2011) doi:10.1111/j.1742-4658.2011.08243.x The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinfor- matics search [Rawlings ND & Bateman A (2009) BMC Genomics 10, 437] revealed that, in seven of  1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hall- mark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to dis- play properties remarkably similar to those of pepsin-like aspartic protein- ases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activ- ity of other aspartic proteinases, and displayed a clear preference for cleav- ing peptide bonds between hydrophobic residues in the P1*P1¢ positions of the substrate. It was completely inhibited by the general inhibitor of aspar- tic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinc- tions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined. Structured digital abstract l Shewasin A cleaves Oxidized Insulin B chain by protease assay (View Interaction 1, 2) Introduction Aspartic proteinases (APs) are widely distributed in nature, including in a variety of infectious organisms, such as Plasmodium falciparum, HIV, and a large num- ber of fungi [1]. However, relatively few have been Abbreviations AP, aspartic proteinase; DABCYL, 4-(dimethylaminoazo)benzene-4-carboxylic acid; DNP, 2,4-dinitrophenyl; E-64, l-trans-epoxysuccinylleucylamide-(4-guanidino)butane; EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; MCA, (7-methoxycoumarin-4-yl) acetic acid; Nbs 2 , 5,5¢-dithio-bis(2-nitrobenzoic acid). FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS 3177 described in bacteria. These enzymes are currently sub- divided into different families and clans as described in the MEROPS database [2]. Seven of these families (A8, A22, A24, A25, A31, A26, and A5) are organized into distinct clans in which, although Asp residues are known to be critical for enzymatic activity, they appear in diverse sequence motifs [2]. Indeed, this set of families contains the only APs of bacterial origin that have so far been characterized: these include sig- nal peptidase II from Escherichia coli, which is the type peptidase of family A8 [3], the prepilin peptidases of family A24 [4], GPR endopeptidase from Bacil- lus megaterium in family A25 [5], omptin from E. coli, which is the type peptidase of family A26 [6], and HybD peptidases of family A31 [7]. The remaining families of APs in the MEROPS database (A1, A2, A3, A9, A11, and A33) all belong to only one clan (AA), with their members being read- ily identified by the presence of characteristic hallmark sequence motifs. These ‘archetypal’ APs include eukaryotic enzymes such as pepsin and viral retropep- sins, including HIV-1 retropepsin. Pepsin-like APs characteristically consist of two internally homologous domains, each of which provides a catalytic Asp to the active site. Each Asp is present in the hallmark motif Asp-Thr ⁄Ser-Gly, followed further downstream by a hydrophobic-hydrophobic-Gly sequence. Together, these motifs form a structural feature known as a psi loop, which serves to locate the two Asp residues nec- essary for operation of the catalytic machinery [1]. In contrast, retroviral-type APs are obligate homodimers, in which each monomer contributes one catalytic motif to one psi loop. Enzymes with a pepsin-like ‘arche- typal’ organization are by far the most numerous and well-characterized APs, and have been thought to be confined to eukaryotes. This has been supported by structural evidence suggesting that pepsin-like enzymes evolved through a gene duplication and fusion event from a retropepsin-type of ancestral gene [8]. However, the absolute requirement for the psi loop structural feature described above provides four landmark motifs (two Asp-Thr ⁄Ser-Gly and two hydrophobic-hydro- phobic-Gly) that are required to be present in con- served locations, and so can be searched for during data mining operations to identify putative pepsin-like APs in any newly sequenced genome. In such an endeavor, contrary to long-held beliefs, pepsin-like APs were detected within the genomes of a few bacte- ria [9]. All of the currently sequenced bacterial genomes ( 1000) were examined, and putative AP- encoding genes were identified in seven species. Of these, two pairs of Asp-Thr ⁄ Ser-Gly + hydrophobic- hydrophobic-Gly motifs were present in the predicted polypeptides from five species, all marine psychro- philes, including Shewanella amazonensis [9]. Prior to this recent report, other publications suggesting the presence of archetypal types of AP in bacteria were somewhat unconvincing [10–12]. Given the potential significance of this detection of AP-encoding genes in a few species of bacteria, it was thus considered of particular importance to produce reliable biochemical data to characterize such putative bacterial gene products and thus establish unequivo- cally whether these encoded protein products were functional enzymes. In this article, we describe the pro- duction of recombinant shewasin A, the pepsin-like homolog from the bacterium S. amazonensis, and dem- onstrate that it displays all of the enzymatic properties characteristic of a eukaryotic pepsin-like AP. We dis- cuss the differences between bacterial and eukaryotic polypeptides, and consider the evolutionary signifi- cance of these observations. Results Expression and purification of recombinant shewasin A In order to characterize one of the bacterial pepsin-like homologs identified by Rawlings & Bateman [9], the gene from S. amazonensis (GenBank: ABL98994.1) was selected. DNA was synthesized (sequence detailed in Fig. S1) to encode the full-length polypeptide, the sequence of which is shown in Fig. 1. The synthetic gene was expressed in E. coli BL21(DE3) as described Fig. 1. Deduced amino acid sequence of S. amazonensis shewa- sin A. The hallmark motifs of pepsin-like APs are highlighted in the sequence, and include: (a) the active site motifs (DT ⁄ SG) (shown in bold in gray boxes); (b) the hydrophobic-hydrophobic-Gly motifs of the psi loops (shown in bold); and (c) the conserved Tyr residue in the ‘flap’ region (double underlined). The eight Cys residues are underlined in the sequence. No signal peptide or propart segment is present in the shewasin A amino acid sequence. The Asp of the active site motif from the N-terminal domain (marked with an aster- isk) was mutated to an Ala to generate the active site mutant shewasin A_(D37A). Although it displays the typical hallmark motifs of pepsin-like APs, shewasin A shows a low overall percentage of sequence identity with eukaryotic pepsin-like enzymes, e.g. pep- sin A (18%), BACE1 (10%), and renin (9%). Characterization of recombinant shewasin A I. Simo˜es et al. 3178 FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS in Experimental procedures, and initial conditions were optimized to enhance the accumulation of recombinant shewasin A, with an N-terminal His-tag, in the soluble fraction of the cell lysates. Metal ion affinity chroma- tography was applied (Fig. 2A), and fractions enriched in shewasin A were pooled and further purified by size-exclusion chromatography on a HiLoad 26 ⁄60 Superdex 200 column (Fig. 2B). SDS ⁄ PAGE analysis of the purified fractions under reducing conditions confirmed the presence of a protein with the predicted molecular mass of 50 kDa (Fig. 2D, lanes 1–4). The identity of this band was further confirmed by western blot analysis with an antibody against His tag (not shown). The purified recombinant shewasin A was subjected to analytical size-exclusion chromatography (Fig. 2C) under nondenaturing conditions and in the absence of a reducing agent, and its molecular mass was deter- mined to be  50 kDa, consistent with the value calcu- lated for the polypeptide (Fig. 1) encoded by the bacterial gene. Thus, recombinant shewasin A exists as a monomeric polypeptide, as observed for the majority of eukaryotic pepsin-like APs studied previously. Shewasin A contains eight Cys residues at noncon- served positions. To evaluate the number of these resi- dues present in a reduced form, a 5,5¢-dithiobis (2-nitrobenzoic acid) (Nbs 2 ) assay was carried out. The number of free thiol groups in recombinant shewa- sin A estimated from Nbs 2 titration was 8.27 ± 0.59. These results clearly suggest that all sulfhydryl groups of shewasin A exist as free thiols, in sharp contrast to its eukaryotic counterparts. Activity and specificity of recombinant shewasin A Recombinant shewasin A was next examined for its ability to cleave a number of polypeptides typically used as AP substrates. Fluorogenic substrates cleaved by renin [(5-[(2-aminoethyl)amino]naphthalene- Fig. 2. Purification and analysis of recombinant shewasin A. Wild-type shewasin A was produced in E. coli in soluble form, fused to an N-terminal His-tag. (A) HisTrapHP chromatogram. Recombinant shewasin A was purified by metal ion affinity chromatography with a HisT- rapHP column. Elution was accomplished by using stepwise increases in concentration of imidazole (50, 100 and 500 m M). The recombinant protein was eluted with 100 m M imidazole, corresponding to fractions highlighted by dotted lines (1 and 2; numbers above the peaks). (B) S200 chromatogram. HisTrap eluate (fractions 1 and 2) was pooled and further purified by size-exclusion chromatography as described in Experimental procedures. Purified recombinant shewasin A (dotted lines, sample 3) was used in subsequent characterization assays. (C) Analytical size-exclusion chromatography of purified recombinant shewasin A. The Superose 12 was equilibrated with 20 m M Hepes buffer (pH 7.5) and 100 m M NaCl. The dots indicate the elution volumes of molecular mass markers used for calibration (from left to right: aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 34 kDa; carbonic anhydrase, 29 kDa; ribonuclease A, 13.7 kDa). The collected fraction is high- lighted by dotted lines, and pooled as fraction 4. (D) SDS ⁄ PAGE analysis of protein fractions collected from the different steps of purification. Lanes 1 and 2: fractions 1 and 2 in (A). Lane 3: fraction 3 in (B). Lane 4: Superose 12 eluate marked with number 4 in (C). The gel was stained with Coomassie Brilliant Blue. I. Simo˜es et al. Characterization of recombinant shewasin A FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS 3179 1-sulfonic acid [EDANS])-Ile-His-Pro-Phe-His-Leu-Val- Ile-His-Thr-Lys(DABCYL)-Arg], HIV-1 retropepsin [Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys- 4-(dimethylaminoazo)benzene-4-carboxylic acid (DAB- CYL)-Arg] and BACE1 [(7-methoxycoumarin-4-yl) acetic acid (MCA)Lys-Ser-Glu-Val-Asn-Leu-Asp-Ala- Glu-Phe-Lys-2,4-dinitrophenyl (DNP)] were not signifi- cantly processed by shewasin A under the conditions tested. The failure to cleave the BACE1 substrate may be noteworthy, in that a phylogenetic analysis of fam- ily A1 members resolved shewasin A into a cluster with BACE1 and its human paralog BACE2, suggest- ing the closest relationship with these eukaryotic enzymes [9]. I n contrast, the fluorogenic peptide (MCA) Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys(DNP), which was originally designed as a substrate for CDR1, an atypical AP from Arabidopsis thaliana [13], was readily hydrolyzed by shewasin A at pH 4. Analy- sis by MS revealed that the primary cleavage site was at Leu*Phe (* indicates the cleavage site), with a fur- ther minor cleavage occurring at the adjacent Phe*Val (Table 1). Incubation of shewasin A with the B chain of oxi- dized insulin at pH 4 was followed by RP-HPLC sepa- ration of the products (not shown) and analysis by MS. For this peptide, two major cleavage sites were identified, Leu15*Tyr16 and Tyr16*Leu17 (Table 1). Thus, shewasin A reveals a specificity that is intrinsic to most eukaryotic pepsin-like APs in cleaving prefer- entially between hydrophobic residues occupying the substrate P1 and P1¢ positions. The final substrate tested was a fluorogenic deriva- tive of the chromogenic peptide Lys-Pro-Ala-Glu- Phe*Nph-Ala-Leu (where Nph is L-norleucine) [14]. The quenched fluorescent version of this peptide, (MCA) Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP), was readily cleaved by shewasin A, displaying typical Michaelis–Menten kinetic behavior. The kinetic parameters determined for cleavage at pH 4.0 were K m = 5.4 lM, k cat = 0.03 s )1 , and k cat ⁄ K m = 5.6 · 10 3 M )1 Æs )1 , respectively. MS analysis revealed that this peptide was preferentially cleaved at Phe*Phe, with a second minor cleavage occurring at Phe*Ala (Table 1). Maximum activity was observed at temperatures between 42 and 50 °C, decreasing so drastically above 50 °C that complete loss of activity was detected at 60 °C (Fig. 3A). The pH dependence of the cleavage of (MCA)Lys- Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP) by recom- binant shewasin A is shown in Fig. 3B. The highest activity was detected at acidic pH values between pH 3.75 and pH 4.5, and only 50% activity was retained at pH 5. At pH 6.0, the enzyme showed no activity towards this substrate (Fig. 3B). This behavior is typical of many eukaryotic APs mainly acting in Table 1. Primary specificity of recombinant shewasin A. Three dif- ferent substrates were incubated with recombinant shewasin A as described in Experimental procedures. The resulting cleavage prod- ucts were identified directly by MS analysis or, in the case of oxi- dized insulin B chain, separated by RP-HPLC prior to identification by MS. Preferential cleavage sites are indicated by (››) and minor cleavage sites by (›). Substrate Sequence CDR1 peptide (MCA)KLHPEVL››F›VLEK(DNP) Oxidized insulin B chain FVNQHLCGSHLVEAL››Y››LVCGERGFFYTPKA Typical peptide (MCA)KKPAEF››F›ALK(DNP) Fig. 3. Effect of temperature and pH on the activity of recombinant shewasin A. Shewasin A was tested for activity with the synthetic fluorogenic peptide (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu- Lys(DNP) as substrate; the chromogenic version of this has been used as a model substrate to characterize pepsin-like enzymes from various sources. (A) Activity studies at different temperatures were performed by incubating shewasin A in 0.05 M sodium ace- tate buffer (pH 4) and 0.1 M NaCl at temperatures between 15 and 60 °C. (B) Activities at different pH values were measured by incu- bating shewasin A at 37 °C with buffers between pH 2.5 and pH 7 containing 0.1 M NaCl (0.05 M sodium citrate, pH 2.5–3.5; 0.05 M sodium acetate, pH 4–5.5, 0.05 M sodium phosphate, pH 6–7). Characterization of recombinant shewasin A I. Simo˜es et al. 3180 FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS acidic environments, including vertebrate pepsins, cathepsin D, and a variety of enzymes of fungal origin [1,15,16], but contrasts with that observed for more specialized APs, which are active at elevated pH values closer to neutrality, e.g. renin and HIV-1 retropepsin. The fact that maximum activity for shewasin A is observed between pH 3.75 and pH 4.5 makes the behavior of the recombinant bacterial AP even more like that of an archetypal pepsin-like enzyme rather than like some of the more ‘specialized’ APs, such as renin and retroviral proteinases. Inhibition and dependence on conserved catalytic residues for shewasin A activity The most frequently applied test employed to classify a newly identified protease is susceptibility to prototypi- cal inhibitors such as pepstatin [1]. Consequently, the effect of pepstatin on the activity of shewasin A was examined; whereas pepstatin completely blocked its proteolytic activity at pH 4, all other inhibitors tested were devoid of inhibitory effect (Table 2). In order to substantiate this finding further, an active site mutant of shewasin A was generated in which the (putative) catalytic Asp of the Asp-Thr-Gly motif of the N-termi- nal domain (Fig. 1) was mutated to an Ala (D37A). This mutant was expressed in E. coli and purified under similar conditions to those used for wild-type shewasin A. Purified shewasin A_(D37A) was analyzed in a size-exclusion chromatography column, and displayed a molecular mass of  50 kDa (Fig. 4A), consistent with that described above for the wild type. Analysis by SDS ⁄ PAGE and western blot with a His-tag antibody (Fig. 4B) revealed that the mutant protein migrated identically to the wild-type shewasin A. In sharp contrast, however, purified shewasin A_(D37A) was completely inactive towards the fluorogenic substrate (MCA)Lys-Lys-Pro- Ala-Glu-Phe-Phe-Ala-Leu- Lys(DNP) at pH 4.0 (Fig. 4C). Discussion Shewasin A exists as a monomer, exhibits activity at acidic pH against a well-documented AP substrate, Table 2. Effect of prototypical proteinase inhibitors on the activity of recombinant shewasin A. Recombinant shewasin A was tested for activity with the synthetic fluorogenic peptide (MCA)Lys-Lys- Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP) as substrate in 0.05 M sodium acetate (pH 4) and 0.1 M NaCl at 37 °C. The enzyme was preincubated in the presence of each prototypical inhibitor for 10 min at 37 °C before substrate addition. Inhibitor Concentration (m M) Activity (%) Pepstatin 0.001 0 Pefabloc 1 87.8 EDTA 5 82.8 E-64 0.01 89.5 Amastatin 0.01 97.2 Bestatin 0.01 92.7 Leupeptin 0.01 91.9 Dithiothreitol 2 91.2 Iodoacetamide 0.05 82.1 Fig. 4. Purification and analysis of recombinant shewasin A active site mutant. The active site mutant shewasin A_(D37A) was expressed in E. coli, purified according to the protocol optimized for shewasin A described in Experimental procedures, and subse- quently analyzed by analytical size-exclusion chromatography in a Superose 12 column (A). (B) Purified shewasin A_(D37A) [fraction delimited by dotted lines in (A)] was analyzed by SDS ⁄ PAGE and western blot (WB) with a His-tag antibody. Wild-type shewasin A (WT) was included for comparison. The gel was stained with Coo- massie Brilliant Blue. (C) Purified recombinant shewasin A_(D37A) was tested for activity with the synthetic fluorogenic peptide (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP) as substrate in 0.05 M sodium acetate buffer (pH 4) and 0.1 M NaCl at 37 °C. I. Simo˜es et al. Characterization of recombinant shewasin A FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS 3181 cleaves its substrates preferentially between hydrophobic amino acids, and is susceptible to inhibition by pepsta- tin. Furthermore, the presence of four typical motifs (two Asp-Thr ⁄ Ser-Gly and two hydrophobic-hydro- phobic-Gly) in its sequence in combination with a total loss of activity as a result of mutation of one of these putative catalytic Asp residues provides further strong evidence that this enzyme is an active pepsin-like AP. To the best of our knowledge, this is the first docu- mentation of such an activity, and establishes beyond doubt that pepsin-like APs belonging to family A1 are not confined to eukaryotes but are encoded in certain species of bacteria. Whereas shewasin A’s enzymatic properties are in good agreement with those of its eukaryotic homo- logs, one obvious molecular feature serves to distin- guish between the bacterial AP and its eukaryotic counterparts. Eukaryotic pepsin-like APs from fam- ily A1 are typically encoded and produced as prep- roenzymes (e.g. pig pepsinogen), consisting of an initial signal peptide, a propart segment, and the mature enzyme region. In sharp contrast, the shewa- sin A polypeptide encoded within the bacterial gen- ome is devoid of both a signal peptide and propart segment (Fig. 1). Eukaryotic AP polypeptides lacking either a signal peptide or a propart segment have been described previously in other species (fungi [17] and oomycetes [18]), but the finding that bacterial APs such as shewasin A lack both of the compo- nents is totally unprecedented. In our studies, recom- binant shewasin A was isolated in an active form directly from the soluble fraction of E. coli cell ly- sates, so the absence of a propart segment would not appear to be detrimental to the folding of this bacterial AP in the heterologous expression system chosen. Further experiments will be necessary to establish the subcellular location and activity of shewasin A within S. amazonensis cells. In eukaryotic zymogens, the propart segment is known to make essential contributions, such as ensur- ing proper folding and intracellular sorting of the zymogen polypeptide, and facilitation of its activation to release the mature enzyme when the appropriate conditions are encountered [1,19]. Interestingly, the presence of the propart segment in proBACE1 was found to have little effect on the intrinsic proteolytic activity as in a typical AP zymogen, but its inclusion at the protein’s N-terminus ensured much more rapid folding of the polypeptide than was observed when only the mature form was produced [20,21]. As BACE1 and BACE2 were predicted to be shewasin A’s closest eukaryotic homologs [9], it is possible that the propart segment in proBACE1 and proBACE2 may represent an ancient version of this domain that might have been acquired throughout proteinase evolution, developing according to evolutionary pressures to extend the lifetime of these eukaryotic APs [22]. Indeed, given the strict requirement of the propart segment for proper folding of the precursors of the large majority of eukaryotic pepsin-like proteinases, active bacterial pepsin homologs lacking the propart segment, such as shewasin A, might represent ‘fossil’ versions of pepsin-like proteinases rather than a derived state resulting from a horizontal gene transfer mechanism. Another interesting feature of the bacterial pepsin homologs [9] is the difference in their Cys content and position within the sequence. Shewasin A contains eight Cys residues (Fig. 1), whereas three, four, seven or eight are present, at nonconserved locations, in the six predicted polypeptides from the other species of bacteria [9]. This contrasts sharply with eukaryotic pepsin-like APs, which commonly contain two, four or six Cys residues located at totally conserved positions, and form one, two or three disulfide bonds, respec- tively. None of the Cys residues in the shewasin A sequence are present at these conserved positions, and their localization along the protein sequence suggests that the Cys residues in this bacterial AP may not form disulfide bonds, as determined from a model of shewasin A built on pig pepsin (data not shown). Determination of shewasin A free sulfhydryl groups by Nbs 2 titration further confirmed this in silico analysis, as all of its eight Cys residues were estimated to exist as free thiols. It is very likely that the Cys residues remain in their reduced form with free SH side chains, which would be consistent with the reducing environ- ment that exists inside bacterial cells. In further support of this interpretation, shewasin A accumulated in a soluble monomeric form in E. coli , and addition of dithiothreitol (at 2 m M) had no effect on either the molecular mass of the active entity or on the activity observed for the purified recombinant wild-type shewasin A (Table 2). A similar result was observed when shewasin A activity was assayed in the presence of iodoacetamide (at 0.05 m M) (Table 2). The absence of a signal peptide at the N-terminus of bacterial APs such as shewasin A also suggests that these might be cytosolic proteins [9]. Accord- ingly, it was expected that shewasin A would be active at pH values reflecting that of the bacterial cytoplasm, i.e. close to neutrality. The presence in shewasin A of an Ala just downstream from the Asp- Thr ⁄ Ser-Gly motif of the C-terminal domain (in the sequence Asp-Ser-Gly-Ala; Fig. 1) was also suggestive of such an effect, because the maximum activity of Characterization of recombinant shewasin A I. Simo˜es et al. 3182 FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS more specialized APs such as renin and HIV-1 retro- pepsin at pH values closer to neutrality has been attributed, at least in part, to the presence of this Ala [23]. This contrasts with the situation in many pepsin- like enzymes, in which the equivalent residue is Thr. However, our experimental observations do not sup- port this interpretation, as shewasin A was shown to be maximally active around pH 4, and no activity whatsoever was detected at pH 6. Thus, this differ- ence in shewasin A catalytic activity from those of other APs with a similar active site sequence motif may be the result of subtle variations in subsite bind- ing pockets. APs of the pepsin-like (A1) family were believed, until recently, to be confined to eukaryotic organisms; our results provide unequivocal experimental substanti- ation that this type of AP is also encoded, but in the form of the mature enzyme, in bacteria. The S. amazonensis pepsin homolog described here is strongly reminiscent of eukaryotic pepsin-like APs. Our findings pose challenges for understanding the evolutionary relationships between bacterial APs and their eukaryotic counterparts, particularly as shewa- sin A was shown to be distantly positioned near the root of the phylogenetic tree derived from family A1 members [9]. The most widely held view has been that retroviral APs represent the ancestral state, and that bilobed pepsin-like proteinases are the result of gene duplication and fusion events [1,8]. As it is now clear that the S. amazonensis genome does encode an active pepsin-like proteinase, it would appear that the gene duplication and fusion may well be very ancient events, preceding the divergence between bacteria and eukaryotes. The recent identification of a novel retroviral-type AP (SpoIIGA) in Bacillus subtilis,a Gram-positive bacterium [24], further contributes to the discussion on the evolutionary relationships between retroviral and pepsin-like APs. This sequence contained one Asp-Thr-Gly motif, consistent with that expected of family A2 members, so that dimerization would be required for activity. Mutational analysis demonstrated the critical role of the Asp for substrate processing; however, attempts at inhibition of the observed activity were rather inconclusive. Further investigations will thus be necessary to demonstrate unequivocally that an ancestral gene encoding a single- lobed AP sequence is present in prokaryotes. However, the evidence currently available does provide an initial indication that the hypothetical gene duplica- tion ⁄ fusion events that may have given rise to the bi-lobed pepsin-like APs, such as shewasin A, might have preceded the most recent common ancestor of prokaryotes and eukaryotes. Experimental procedures Cloning of S. amazonensis gene encoding shewasin A DNA encoding the S. amazonensis pepsin homolog gene (gene locus Sama_0787; genomic sequence available at the EBI Data Bank under the accession number ABL98994) was chemically synthesized (Genscript, Piscataway, NJ, USA), and optimized for codon usage in E. coli to enhance protein expression. The synthetic gene sequence (detailed in Fig. S1) included restriction sites for NdeI and XhoI at the 5¢-end and 3¢-end, respectively, to facilitate subsequent subcloning in pET28a (Novagen, Gibbstown, NJ, USA) in-frame with an N-terminal His-tag. The positive clones selected by restriction analysis were confirmed by DNA sequencing. The QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to generate the active site mutant shewasin A_(D37A) in the vector pET28a, using the primers 5¢-AGCGTGAACCTGATTATT GCGA CCGGCAGCAGCACCCTG-3¢ (forward primer) and 5¢-CAGGGTGCTGCTGCCGGT CGCAATAATCAGGTT CACGCT-3¢ (reverse primer) (mutation sites underlined). The positive mutant clones were confirmed by DNA sequencing. Expression and purification of recombinant shewasin A and the active site mutant in E. coli Wild-type shewasin A and shewasin A_(D37A) were trans- formed into E. coli BL21(DE3). The method of recombi- nant protein expression was optimized to maximize the yield of protein in soluble form, and the resulting condi- tions were used in all subsequent experiments as follows. After growth of the cells at 30 °CtoD 600 nm of 0.6, gene expression was induced by the addition of isopropyl thio-b- D-galactoside (0.05 mM final concentration). After 4 h at 30 °C, cells were harvested by centrifugation at 8983 g at 4 °C for 20 min, resuspended in 20 m M sodium phosphate buffer (pH 7.4) containing 10 m M imidazole and 0.5 M NaCl (binding buffer for immobilized metal ion affinity chromatography), and lysed with lysozyme (100 lgÆmL )1 ). After freezing and thawing, DNase (100 lgÆmL )1 ) and MgCl 2 (100 mM) were added, and the reaction mixture was incubated for 2 h at 4 °C. The cell lysate was centrifuged at 12 000 g and 4 °C for 12 min. The soluble fraction was fil- tered through 0.2-lm filters, and immediately loaded onto a HisTrapHP 5-mL column (GE Healthcare Life Sciences, Uppsala, Sweden) previously equilibrated in binding buffer. After sample loading, the column was connected to an FPLC system (DuoFlow-BioRad, Hercules, CA, USA), and extensively washed with binding buffer until A 280 nm reached a steady baseline. Protein elution was carried out by increasing the concentration of imidazole stepwise I. Simo˜es et al. Characterization of recombinant shewasin A FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS 3183 (50, 100 and 500 mM) in the same buffer. Both shewasin A and shewasin A_(D37A) were eluted with the buffer con- taining 100 m M imidazole. Pooled fractions were applied to a HiLoad 26 ⁄ 60 Superdex 200 gel filtration column (GE Healthcare Life Sciences) connected to an FPLC system (DuoFlow-BioRad) equilibrated in 20 m M Hepes buffer (pH 7.5) containing 100 m M NaCl for further purification and imidazole removal. Size-exclusion chromatography The molecular masses of purified recombinant shewasin A and shewasin A_(D37A) were estimated under nondenatur- ing conditions by size-exclusion chromatography on a Superose 12 (GE Healthcare Life Sciences) column con- nected to an FPLC system (DuoFlow-BioRad). The column was equilibrated in 20 m M Hepes buffer (pH 7.5) contain- ing 100 m M NaCl, and calibrated with Gel Filtration LMW and HMW calibration kits (GE Healthcare Life Sciences), according to the manufacturer’s instructions. The molecular mass markers used for calibration were aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhy- drase (29 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa). Nbs 2 assay The sulfhydryl contents of recombinant shewasin A were determined spectrophotometrically at 412 nm with Nbs 2 [25]. Purified recombinant shewasin A (0.37 lM) was incu- bated at 30 °C with 3.3 m M Nbs 2 in 0.1 M sodium phos- phate buffer (pH 8.0) containing 1 m M EDTA. A control without enzyme was performed to measure the spontaneous breakdown of the reagent, and this value was used to cor- rect the titration value obtained for recombinant shewa- sin A. The number of sulfhydryl groups was calculated by using the molar extinction coefficient of 2-nitro-5-thioben- zoic acid (14 150 M )1 Æcm )1 ). Enzyme assays The proteolytic activities of purified recombinant shewa- sin A and shewasin A_(D37A) were tested against several fluorogenic peptides, initially at concentrations between 1 and 2 l M in buffers at different pH values containing 0.1 M NaCl and 8% (v ⁄ v) dimethylsulfoxide. These included the renin substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu- Val-Ile-His-Thr-Lys(DABCYL)-Arg and the HIV-1 retropepsin protease substrate Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr- Pro-Ile-Val-Gln-Lys(DABCYL)-Arg, both from Sigma (St Louis, MO, USA), and the BACE1 substrate (MCA)Lys- Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DNP), as well as (MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys- (DNP) and (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu- Lys(DNP), all synthesized by Genosphere Biotechnologies (Paris, France). The last of these peptides was found to be cleaved readily, and the rate of hydrolysis was monitored at an excitation wavelength of 328 nm and an emission wave- length of 393 nm. The relationship between fluorescence change and peptide concentration was calculated by mea- suring the total fluorescence change that occurred upon complete hydrolysis of the peptide. Kinetic parameters for the cleavage reaction were calculated from the Lineweaver– Burk plot with appropriate software. For activity studies at different pH values, the following buffers, all containing 0.1 M NaCl, were used between pH 2.5 and pH 7 at 37 °C: 0.05 M sodium citrate (pH 2.5–3.5); 0.05 M sodium acetate (pH 4–5.5); and 0.05 M sodium phosphate (pH 6–7). For activity studies at different temperatures, recombinant shewasin A was incubated in 0.05 M sodium acetate and 0.1 M NaCl (pH 4) at temperatures between 15 and 60 °C. The effects of various inhibitors on the proteolytic activity of shewasin A were assayed by preincubating the enzyme with each compound for 10 min at 37 °C in 0.05 M sodium acetate buffer (pH 4.0) containing 0.1 M NaCl before deter- mination of the residual proteolytic activity. Shewa- sin A_(D37A) was examined for activity under the same assay conditions. Digestion of oxidized insulin B chain Digestion of oxidized insulin B chain by purified recombi- nant shewasin A was carried out for 4 h at 37 ° C in 0.1 M sodium acetate buffer (pH 4). The reaction was stopped with 0.6% (v ⁄ v) trifluoroacetic acid (final concentration) and, after centrifugation (12 000 g, 5 min), digestion frag- ments were separated by RP-HPLC on a C18 column, using a Prominence system (Shimadzu Corporation, Tokyo, Japan) with a KROMASIL 100 C18 250 · 4.6 mm column. Elution was carried out with a linear gradient of acetoni- trile (0–80%) in 0.1% (v ⁄ v) trifluoroacetic acid for 30 min at a flow rate of 1 mLÆ min )1 . Absorbance was monitored at 220 nm, and the isolated peptides were collected, freeze- dried, and submitted to identification with a 4000 QTRAP system (Proteomics Unit of the Center for Neuroscience and Cell Biology, University of Coimbra, Portugal). PAGE and immunoblotting Protein samples were separated by SDS ⁄ PAGE, using 12% gels in a Bio-Rad Mini Protean III electrophoresis appara- tus (Bio-Rad, Hercules, CA, USA). Gels were stained with Coomassie Brilliant Blue R-250 (Sigma). For immunoblotting analysis, protein samples were separated by SDS ⁄ PAGE (12% gels) and transferred to a poly(vinylidene difluoride) membrane for immunoblotting (40 V, overnight, at 10 °C). The membranes were blocked for 60 min with 5% (w ⁄ v) nonfat dry milk plus 0.1% (v ⁄ v) Tween-20 in NaCl ⁄ Tris, and then incubated at room temperature for 60 min with the primary antibody, mouse His-tag antibody (GenScript; Characterization of recombinant shewasin A I. Simo˜es et al. 3184 FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS 1 : 5000 dilution). After several washes with 0.5% (w ⁄ v) nonfat dry milk plus 0.1% (v ⁄ v) Tween-20 in NaCl ⁄ Tris, the membranes were incubated at room temperature for 60 min with secondary antibody [alkaline phosphatase-con- jugated goat anti-(mouse IgG+ IgM)] (GE Healthcare; 1 : 10 000 dilution). The membranes were again washed, and alkaline phosphatase activity was visualized by the enhanced chemifluorescence method using ECF substrate (GE Healthcare) on a Molecular Imager FX System (Bio-Rad). 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Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than miss- ing files) should be addressed to the authors. Characterization of recombinant shewasin A I. Simo˜es et al. 3186 FEBS Journal 278 (2011) 3177–3186 ª 2011 The Authors Journal compilation ª 2011 FEBS . Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis Isaura Simo ˜ es 1,2 , Rosa ´ rio Faro 1 , Daniel Bur 3 , John Kay 4 and. pro- duction of recombinant shewasin A, the pepsin- like homolog from the bacterium S. amazonensis, and dem- onstrate that it displays all of the enzymatic properties characteristic