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

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 e

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis

Isaura Simo˜es1,2, Rosa´rio Faro1, Daniel Bur3, John Kay4and Carlos Faro1,2

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 catalyaspar-tic 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 Shewanellagene 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; Nbs2, 5,5¢-dithio-bis(2-nitrobenzoic acid).

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 bactebacte-rial

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 pepshewa-sin-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%).

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) (Nbs2) assay was carried out The number of free thiol groups in recombinant shewa-sin A estimated from Nbs2 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.

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] In 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

Km= 5.4 lM, kcat= 0.03 s)1, and kcat⁄ Km= 5.6·

103M )1Æs)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 50C, decreasing so drastically above

50C that complete loss of activity was detected at

60C (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 (›).

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).

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.

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.

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 foldensur-ing and intracellular sortensur-ing 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 Nbs2 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 mM) 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 mM) (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

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

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¢-AGCGTGAACCTGATTATTGCGA

5¢-CAGGGTGCTGCTGCCGGTCGCAATAATCAGGTT 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 30C to D600 nmof 0.6, gene expression was induced by the addition of isopropyl

thio-b-D-galactoside (0.05 mM final concentration) After 4 h at

30C, cells were harvested by centrifugation at 8983 g at

NaCl (binding buffer for immobilized metal ion affinity chromatography), and lysed with lysozyme (100 lgÆmL)1)

MgCl2(100 mM) were added, and the reaction mixture was incubated for 2 h at 4C The cell lysate was centrifuged at

12 000 g and 4C 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

reached a steady baseline Protein elution was carried out

by increasing the concentration of imidazole stepwise

(50, 100 and 500 mM) in the same buffer Both shewasin A

and shewasin A_(D37A) were eluted with the buffer

con-taining 100 mMimidazole Pooled fractions were applied to

Healthcare Life Sciences) connected to an FPLC system

(pH 7.5) containing 100 mM 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 mMHepes buffer (pH 7.5)

contain-ing 100 mMNaCl, 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)

Nbs2assay

The sulfhydryl contents of recombinant shewasin A were

[25] Purified recombinant shewasin A (0.37 lM) was

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)1Æcm)1)

Enzyme assays

The proteolytic activities of purified recombinant

shewa-sin A and shewashewa-sin A_(D37A) were tested against several

fluorogenic peptides, initially at concentrations between 1

and 2 lMin buffers at different pH values containing 0.1M

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

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

0.05M sodium citrate (pH 2.5–3.5); 0.05Msodium acetate

activity studies at different temperatures, recombinant

The effects of various inhibitors on the proteolytic activity

of shewasin A were assayed by preincubating the enzyme

acetate buffer (pH 4.0) containing 0.1MNaCl before

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 37C in 0.1M

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,

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 mLÆmin)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

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

(12% gels) and transferred to a poly(vinylidene difluoride) membrane for immunoblotting (40 V, overnight, at 10C)

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;

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)

Acknowledgements

MS analysis was performed in the Proteomic Facility of

the Center for Neuroscience and Cell biology (CNC)

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Supporting information

The following supplementary material is available:

Fig S1 Nucleotide sequence of codon-optimized

she-wasin A gene and deduced amino acid sequence

This supplementary material can be found in the online version of this article

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