Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

As chemoheterotrophs, fungi acquire all their nutrients from the environment and as a result have evolved a battery of hydrolases capable of degrading most biopolymers that they encounter in nature. The genes encoding these enzymes are regulated by the presence or absence of specific inducers, with examples including cellulase induction by cellulose (Murray et al., 2003), amylase induction by (iso)maltose (Kato et al., 2002), and peptidase induction by peptides and proteins (Polaina & MacCabe, 2007). The secreted xylanolytic (Polizeli et al., 2005), cellulolytic (Stricker et al., 2008) and proteolytic systems (Kredics et al., 2005) in fungi have been well characterized due to their relative ease of purification and high production yields. From studies to date it is clear that in addition to endo-acting secreted peptidases, fungi possess numerous intracellular peptidases with a range of substrate specificities that complete protein degradation.

Talaromyces emersonii is a thermophilic filamentous fungus that inhabits soil, decaying masses of plant material, and other accumulations of organic matter wherein the warm, humid and aerobic environment provides the basic conditions for its development (Allen & Emerson, 1949). It has generally recognized as safe (GRAS) status, making it a biotechnologically relevant organism to rival Aspergillus and Trichoderma species for the production of important industrial enzymes. T. emersonii has been shown to secrete a wide array of hydrolytic enzymes under defined growth conditions, and possesses complete xylan- and cellulase-degrading systems that consist of diversified carbohydrases (Heneghan et al., 2007; Murray et al., 2001, 2003; Reen et al., 2003; Tuohy et al., 1993, 1994, 2002). Previously, we have shown that the presence of peptides in growth media induces expression of an extracellular peptidase with broad substrate specificity in T. emersonii (O'Donoghue et al., 2008). In this study we examine intracellular proteolytic activity in T. emersonii under similar growth conditions. In order to better understand the role of intracellular peptidases we describe the purification and classification of the enzyme with the highest detectable activity, a prolyl aminopeptidase (PAP).

PAPs are widespread in nature but little is known about their biological function. The unique structure of proline prevents its cleavage by many broad-specificity aminopeptidases (Cunningham & O'Connor, 1997). Accordingly, the need to hydrolyse such bonds has led to the evolution of a specific subset of enzymes (PAPs) capable of cleaving N-terminal proline. Many bacteria and fungi that produce PAPs are pathogenic. A role for PAP in infectivity has been reported (Zhang et al., 2007), and it has also been proposed as a viable drug target (Felipe et al., 2005). Proline, the product of PAP, has multiple physiological functions in micro-organisms, plants and animals, and its accumulation has been implicated in stress responses in yeast and in the suppression of apoptosis in fungi (Takagi, 2008). PAP is an important enzyme industrially, as fungi that possess intracellular proline aminopeptidase-type activity have been extensively used in flavour development of food products (Bolumar et al., 2003; Fuke & Matsuoka, 1993).

To date, characterization of PAPs has mainly been carried out with enzymes from bacterial sources (Supplementary Table S3). A number of PAP-type activities in fungi have been reported (Bolumar et al., 2003; Fuke & Matsuoka, 1993; Hiwatashi et al., 2004), but no corresponding protein sequences have been assigned for these enzymes. However, more recently, one study by Basten et al. (2005) identified a gene encoding PapA from Aspergillus niger and characterized the recombinant protein.

This work describes the purification and characterization of a native fungal PAP. Cloning and sequence analysis of its gene and interrogation of genome databases allowed the identification of many additional homologous members of this unusual family of peptidases. Furthermore, we show that mRNA levels are regulated by the composition of the growth medium and propose a direct link between extracellular peptidases that yield proline-capped peptides and the intracellular uncapping activity of TePAP.

All chemicals and reagents were obtained from Sigma–Aldrich unless otherwise stated.

Fungal growth.
T. emersonii was obtained from laboratory stocks and routinely subcultured on Sabouraud dextrose agar (SDA) plates at 45 °C. Mycelial plugs from SDA plates were used to inoculate liquid medium for peptidase production that contained 0.5 % (w/v) yeast nitrogen base without amino acids and ammonium sulfate (YNB), supplemented with 2 % (w/v) glucose or glycerol and 0.5 % (w/v) casein, casitone (BD Biosciences), Casamino acids (BD Biosciences) or ammonium sulfate, as indicated. All liquid cultures were grown at 45 °C on an orbital shaker at 220 r.p.m.

Preparation of fungal extract and peptidase screen.
Mycelia were harvested, frozen in liquid N₂ and disrupted by grinding with a mortar and pestle in the presence of glass beads. The resultant biomass was resuspended in buffer A (0.05 M Tris/HCl, pH 7.4), containing 200 µM DTT, 0.001 % (v/v) Tween 80 and 5 U DNase (Roche Applied Science), and incubated overnight at 4 °C with gentle mixing. Glass beads and cellular debris were removed by centrifugation at 15 000 g for 30 min and the supernatant was filtered through a 0.22 µm pore-size filter (Millipore). Peptidase activity was assessed using 1 mM p-nitroanilide (pNA) peptides listed in Supplementary Table S1. Assays were performed in 0.3 M Na₂HPO₄/NaH₂PO₄, pH 7.4, for 30 min at 50 °C. The reaction was terminated by the addition of an equal volume of 30 % (v/v) acetic acid and absorbance was measured using a Wallac Victor multilabel counter at 405 nm. Three replicates of each test and control were assayed and appropriate enzyme, substrate and reagent controls were included.

Enzyme purification.
For protein purification, T. emersonii was cultured for 72 h in medium containing glucose and casitone. Mycelia were harvested as outlined above, and the intracellular extract was concentrated and dialysed against buffer A in a stirred ultrafiltration cell fitted with a 10 kDa membrane (Millipore). Protein present in the crude extract was recovered by centrifugation at 15 000 g for 30 min following sequential precipitation with 45 and 85 % (w/v) ammonium sulfate. The final precipitate was resuspended in buffer A and desalted on a Sephadex G25 column. Proteolytically active fractions were applied to a Phenyl Sepharose CL-4B column and protein was eluted using a linear gradient of 0–5 % ethylene glycol in buffer A. Active fractions were pooled, adjusted to 0.2 M NaCl, reapplied to the column and eluted in a decreasing salt gradient (0.2–0 M NaCl). Enzyme-active fractions were pooled and dialysed against buffer A and applied to a DEAE-Sepharose CL-6B column. Adsorbed protein was eluted in a gradient of 0–0.35 M NaCl, concentrated in a Centricon 10 (Millipore) and applied to a Sephacryl S-300 HR column. Protein was eluted in buffer A containing 0.1 M NaCl. The protein concentration in chromatography fractions was measured routinely by determining A₂₈₀, while pooled fractions were quantified using a modified Lowry method (Bensadoun & Weinstein, 1976) standardized against BSA fraction V. A molar absorption coefficient of 8800 M^–1 cm^–1 was used to calculate activity against P-pNA (Varmanen et al., 1996).

Enzyme characterization.
TePAP specificity was analysed using peptidyl-pNA substrates as outlined above. All assays were performed in triplicate and activity was calculated relative to P-pNA. Optimal pH for TePAP activity was determined using the following buffer solutions: McIlvaine buffer (pH 3.8–6.4), sodium phosphate buffer (pH 6.6–7.8), Tris/HCl buffer (pH 8–9.2) and sodium carbonate/bicarbonate buffer (pH 9.2–10.4). The optimum temperature for activity was determined over a range from 20 to 90 °C. Temperature stability was determined by pre-incubating purified TePAP for 30 min at varying temperatures. All temperature assays were performed at pH 7.4 and enzyme stored at 4 °C served as a stability control. The effects of peptidase inhibitors, amino acid-modifying reagents, metal ions and chelators on purified TePAP were analysed using P-pNA, following pre-incubation for 10 min with the target reagent at room temperature (Table 1b).

Determination of kinetic parameters.
Activity of TePAP (1 nM) was monitored using L-proline-7-amino-4-methylcoumarin (Pro-AMC) as substrate in a Wallac Victor multilabel counter (Perkin Elmer) set at excitation (λ_ex) 360 nm and emission (λ_em) 440 nm. Kinetic constants, K_m and V_max were calculated by nonlinear regression using KaleidaGraph (Synergy Software), following continuous assay at pH 7.4 and 45 °C with 1–150 µM Pro-AMC.

Protein characterization.
Protein purity and molecular mass were evaluated by reducing SDS-PAGE in a 10 % Tris-glycine gel stained with 0.02 % (w/v) Coomassie brilliant blue G-250. The protein band was excised from the gel and trypsin-digested, and internal peptide sequences were determined by liquid chromatography (LC)-MS/MS (Jiménez et al., 2001) using a Micromass QTOF2 mass spectrometer (at the University of Nottingham, UK). Zymography was performed by native PAGE in an 8 % Tris-glycine gel and incubated in 100 µM Pro-AMC, 0.3 M Tris/HCl, pH 7.2, at 50 °C for 5 min (Yasothornsrikul & Hook, 2000). Active enzyme was visualized using a Fluor-S Multimager (Bio-Rad).

DNA extraction and cloning of TePAP.
DNA was isolated (Raeder & Broda, 1985) and RNase-treated. A partial gene sequence encoding TePAP was PCR-amplified from genomic DNA using degenerate primers (5'-TTRCARGGNGGNCCNGG-3' and 5'-CCNCCRAARCTYTGNCC-3', where N=A, C, T or G; Y=C or T; and R=A or G) designed from highly conserved regions of fungal PAPs (GenBank accession numbers CAC40647 and CAC40648). The PCR product was subcloned into the pGEM-T Easy vector (Promega), propagated in E. coli JM109 cells and sequenced. A DIG-labelled DNA probe (Roche Applied Science) was generated using gene-specific oligonucleotides (5'-ATCCTCTTCCTCGACCAACGCGG-3' and 5'-AATCCGCCGAAACTCTGCCCGA-3'). This probe was used to screen a T. emersonii genomic library (Grassick et al., 2004) for bacteriophage containing the full-length Tepap gene. Positive clones were sequenced using 5'-CCGCGTTGGTCGAGGAAGAGGAT-3' and 5'-TCGGGCAGAGTTTCGGCGGATT-3' oligonucleotides.

RNA extraction and Northern blot analysis.
Total RNA was isolated from T. emersonii with TRI-Reagent (Molecular Research Centre). RNA was treated with DNase to remove residual genomic DNA and converted to cDNA using M-MLV reverse transcriptase (Applied Biosystems). The complete coding sequence of Tepap was confirmed by PCR amplification and sequencing of cDNA using gene-specific oligonucleotides (5'-TCCTAGTTCTGCGGGTCAGAC-3' and 5'-TGTTCTGGACTGGAGACTATG-3'). Northern blot analyses were performed on total RNA as outlined previously (Murray et al., 2003), using the DIG-labelled Tepap probe and detection with anti-DIG alkaline phosphatase antibody (Roche Molecular Biochemicals). Membranes were stripped in 0.2 M Tris/HCl, pH 7.5, 50 % (v/v) formamide at 80 °C and hybridized with DIG-labelled 18S rRNA probe (GenBank accession no. D88321.2). Images were visualized using a Fluor-S MultiImager (Bio-Rad).

Sequence and phylogenetic analyses.
Sequences of known, biochemically characterized PAP enzymes from the BRENDA database () were retrieved using the Universal Protein Resource (UniProt, 2007). CLUSTAL_X was used to align protein sequences (), and sequences were manually modified where required. CLUSTAL_X was also used to construct an unrooted phylogenetic tree by the neighbour-joining method, and 1000 bootstrap trials were used (Larkin et al., 2007). The tree was displayed with TreeView. To identify putative fungal PAPs, TePAP was used to query completed fungal genomes available at , and . BLASTP analysis was used to identify other protein sequences with significant homology, while secondary structure was predicted by PSIPRED (McGuffin et al., 2000).

Intracellular peptidase screen
The effects of medium composition on intracellular peptidase activities in T. emersonii mycelial lysates was assessed following 72 h growth and results are summarized in Supplementary Table S1. Hydrolysis of Suc-Ala-Ala-Pro-Phe-pNA (Suc-AAPF-pNA), Suc-Ala-Ala-Ala-pNa (Suc-AAA-pNA), P-pNA, Lys-pNA (K-pNA) and Phe-pNA (F-pNA) was observed under all culture conditions tested; activities against all other substrates were ≤1 IU mg^–1 (Supplementary Table S1). Activity against P-pNA was the predominant activity in mycelia and was maximal in cells grown in a combination of glucose and casitone. Levels of the other two mono-aminopeptidase-type activities and the chymotrypsin-type activity (Suc-AAPF-pNA) were lower under all conditions tested. Moderate levels of the keratinase-type activity (Suc-AAA-pNA) were detected.

TePAP purification
P-pNA activity in mycelia increased with time over a 60 h growth period after inoculation, and maximum activity was observed between 60 and 84 h of growth (data not shown). Extracellular P–pNA activity was not detected under these conditions (data not shown). Purification of a PAP was therefore carried out from a crude extract prepared from T. emersonii mycelia harvested after 72 h growth in medium containing glucose and casitone. A homogeneous enzyme preparation (TePAP) was purified 78-fold by ammonium sulfate fractionation, followed by five successive chromatographic steps to a final yield of 0.6 %, as outlined in Supplementary Table S2. The estimated M_r of 270 000 for the native enzyme was determined by both gel filtration chromatography and zymography (Fig. 1). The observation of a single protein band with an M_r of 46 000 following SDS–PAGE suggests that the enzyme exists in its native state as a homo-hexamer.

(23K): Fig. 1. Purification of TePAP by size-exclusion chromatography using Sephacryl S-300 HR. Activity (black line) and protein abundance (grey line) are indicated. Arrows correspond to the peak fractions for gel filtration standards: ferritin, 440 000 Da; catalase, 240 000 Da; amylase, 200 000 Da. Fractions 68–78 were pooled and analysed by reducing SDS-PAGE followed by Coomassie staining (lane 2), and by native PAGE followed by zymography using Pro-AMC as substrate (lane 4). Denatured and native molecular mass markers are shown in lanes 1 and 3, respectively.

Enzymic characterization of TePAP
TePAP displayed 50-fold greater specificity towards P-pNA compared with Ala-pNA (A-pNA) and Val-pNA (V-pNA), and ≥100 fold greater specificity compared with all other aminopeptidase substrates tested (Table 1). No activity was detected against di- and tri-peptidyl pNA substrates. In addition, purified TePAP was shown to be incapable of cleaving K-pNA, F-pNA, Suc-AAPF-pNA or Suc-AAA-pNA, indicating that it was not responsible for the other peptidase activities detected in T. emersonii mycelial lysates. TePAP was most active against P-pNA between pH 7 and pH 8, with the highest activity observed at pH 7.4 (Fig. 2). At pH 7.4, the optimal temperature for activity was 50 °C, but activity dropped rapidly above this temperature with only 50 % remaining at 60 °C. Under optimal assay conditions (pH 7.4, 50 °C), TePAP retained ∼40 % activity after 1 h and was completely inactivated after 2 h. Under optimal pH conditions, K_m, k_cat and k_cat/K_m values of 101 µM, 213.7 s^–1 and 2.1x10⁶ M^–1 s^–1 respectively, were obtained using a fluorescent Pro-AMC substrate.

Table 1. (a) Substrate specificity and (b) inhibition profile of purified TePAP (a) Relative activity was expressed as a percentage of P-pNA activity. No activity was detected against K-pNA, E-pNA, M-pNA, AAP-pNA, Suc-AAA-pNA and Suc-AAPF-pNA. (b) Greater than 90 % activity was observed in the presence of MgSO4, MnSO4, MnCl2, LiSO4, CdCl2, KI, ZnSO4, CaCl2, 1,10-phenanthroline, pepstatin, bestatin, leupeptin, EDTA, AEBSF and E-64. All assays were carried out in triplicate and are expressed as mean±SEM.

(12K): Fig. 2. Effect of temperature and pH on the activity and stability of purified TePAP. (a) Effect of pH on TePAP activity. (b) Effect of temperature on TePAP activity. (c) Effect of temperature on TePAP stability at 4 °C (broken line), 50 °C (solid line) and 60 °C (dotted line). P-pNA was used for all assays. For assays see Methods.

Inhibition of TePAP activity
The effects of amino-acid-modifying reagents and class-specific peptidase inhibitors on TePAP activity are shown in Table 1. Bestatin, a specific inhibitor of aminopeptidases, had no effect on activity, nor did the serine and aspartic peptidase inhibitors 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and pepstatin, respectively. The histidine-modifying reagent diethylpyrocarbonate (DEPC) caused a 97 % reduction in enzyme activity. Cysteine peptidase inhibitors such as p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM), iodoacetic acid and HgCl₂ all inhibited P-pNA hydrolysis to varying extents, although E-64 [trans-epoxy-succinyl-L-leucylamido-(4-guanidino)-butane] did not. The serine/cysteine inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibited TePAP moderately. No effect on activity was detected in the presence of a range of metal salts or the chelators EDTA and 1,10–phenanthroline, suggesting that TePAP activity is not metal-dependent.

Cloning and sequence analysis of Tepap
Peptide sequencing of TePAP revealed that the enzyme is related to Aspergillus niger PapA and displays homology to a number of putative and unassigned fungal sequences (Fig. 3a). Degenerate oligonucleotides were designed from regions of homology that flank the TePAP peptides in other PAPs. These oligonucleotides facilitated PCR amplification and cloning of a 407 bp fragment from T. emersonii genomic DNA encoding Tepap. This sequence was used to design a specific DNA probe to screen a T. emersonii genomic library. The Tepap gene and protein-coding region were obtained by sequencing of a genomic clone and cDNA, respectively (GenBank accession no. AF439997). The cDNA sequence of Tepap is composed of nine exons, which encode a polypeptide of 448 amino acids with a predicted molecular mass of 50.6 kDa. Signal P (Bendtsen et al., 2004) analysis determined that TePAP does not possess a secretory signal peptide. Conserved domain analysis of the sequence revealed it to be an α/β hydrolase fold serine peptidase containing a Ser-Asp-His active site triad. The nucleophilic serine is in the Gly-X-Ser-B-Z motif common to serine peptidases, where X represents any amino acid, and B and Z represent amino acids with a bulky R group and a small R group, respectively (Fig. 3b).

(87K): Fig. 3. Sequence analysis of TePAP. (a) Protein alignment of TePAP with known bacterial and fungal PAPs from Aspergillus niger, PapA; Aeromonas sobria, AsPAP; and X. campestris, XcPIP. Amino acids conserved in TePAP are highlighted in grey. Predicted helices (cylinders) and strands (arrows) in multimeric PAPs corresponding to confirmed elements in XcPIP are shown in dark grey. Predicted helices unique to all multimeric PAPs are shown in light grey. Peptide sequences determined by liquid chromatography-MS/MS are underlined. Regions used to design degenerate oligonucleotides are indicated with dashed arrows. (b) Catalytically and functionally important residues in PAPs. Residues with important functions have been deciphered from structural studies with XcPIP. These residues are listed in (b) and are indicated by and * in (a). The (semi)-conservation of these residues in the four aligned PAPs from (a) is indicated.

Expression profile of Tepap
Total RNA was extracted from T. emersonii after 24 and 48 h growth in a variety of media, as outlined in Fig. 4. Transcript levels of Tepap were determined by hybridization with a DNA probe specific to exon 3. These studies showed that low levels of mRNA were detectable after 24 h growth under all conditions examined. However, Tepap mRNA accumulated after a further 24 h growth in medium containing both glucose and casitone, but not when either compound was present alone. Substitution of glycerol for glucose failed to reproduce the expression levels seen in glucose+casitone medium. Addition of an easily metabolized nitrogen source such as ammonium sulfate had no effect on Tepap expression levels.

(24K): Fig. 4. Northern analysis of total RNA for Tepap extracted from T. emersonii.

Sequence analysis and secondary structure prediction
Analysis of enzymes in the BRENDA database yielded 30 PAPs that have been biochemically characterized. As of May 2009, 12 of these enzymes had been assigned protein sequences while we retrieved a further seven sequences by homology from available genomic sequences (see Supplementary Table S3). As well as being capable of cleaving Pro bonds, all PAPs were described as being sensitive to one or more thiol-modifying reagent(s). The subunit number of the native form of each enzyme was also recorded. Aligning TePAP with a representative monomeric bacterial (Xanthomonas campestris, XcPIP), a multimeric bacterial (Aeromonas sobria, AsPAP) and a multimeric fungal (Aspergillus niger, PapA) enzyme showed specific regions of high homology, but overall sequence identities of the full-length proteins were 13, 40 and 72 % respectively (Fig. 3a). A secondary structure prediction of each protein identified 12 α-helices and eight β-strands that correlate with the known structure of XcPIP (Medrano et al., 1998). An additional four helices (Mα1–4) are present in TePAP, PapA and AsPAP, and are predicted to be unique to the multimeric PAPs.

A phylogenetic tree of all 19 available PAP sequences from biochemically characterized enzymes was constructed. The tree revealed the presence of two major subfamilies that are distinguishable by the presence or absence of Mα1–4 (Fig. 5). Interestingly, the MEROPS database has grouped PAPs into two subfamilies on the basis of similarity to Neisseria gonorrhoeae PAP (S33.001) or AsPAP (S33.008). We define here that this classification correlates exactly with the presence of four additional helices (Mα1–4) in all S33.008 subfamily proteins and their absence in S33.001 enzymes. S33.008 proteins are found in eukaryotes (fungi and plants) and some bacteria, while S33.001 enzymes are only found in bacteria.

(24K): Fig. 5. Phylogenetic tree of characterized PAPs from all known organisms. The tree was constructed using a CLUSTAL_X alignment of entire PAP protein sequences. Asterisks indicate S33.001 proteins that are multimeric. The branch lengths are proportional to sequence divergence and the scale of 0.1 amino acid changes per site is indicated in the bottom left. The numbers displayed are bootstrap values (1000 replicates). See Supplementary Table S3 for the full unabbreviated names.

BLASTP searches performed with TePAP revealed hypothetical proteins in the genomes of many fungi. The sequences closest to TePAP were identified in Aspergillus fumigatus (71 % identity), Neosartorya fischeri (71 %) and Aspergillus clavatus (70 %). Matching sequences were also present in model fungal species (e.g. Neurospora crassa, Aspergillus nidulans), human pathogens (e.g. Candida albicans, Paracoccoidis brasiliensis) and plant pathogens (e.g. Fusarium graminearum, Magnaporthe grisea). These proteins were aligned based on the presence of the putative catalytic serine, aspartic and histidine residues identified by homology (Supplementary Table S4). Notable fungal genomes lacking PAP homologues are listed in the legend of Supplementary Table S4. Fungi have the ability to digest polymeric compounds encountered in their environment into smaller constituents that are readily taken up by mycelia as carbon and nitrogen nutrient sources. This characteristic is reflected in the high levels of hydrolytic enzyme activities that have been found in both the mycelial lysates and culture filtrates of many fungi. Due to their opportunistic ecological niche, saprophytes such as T. emersonii have the ability to utilize a variety of biopolymers and therefore display a wide range of enzymic activities. Proteins and peptides are the most abundant naturally occurring nitrogen sources and due to protein sequence heterogeneity, a range of peptidases with varying specificities are required to carry out the degradation of these proteins and peptides into smaller more readily assimilated peptides.

Previous work has shown that peptide hydrolysates such as casitone (an enzymic hydrolysate of casein) are excellent inducers of a major extracellular peptidase in T. emersonii (O'Donoghue et al., 2008). To expand our knowledge of the spectrum of peptidases produced by this fungus, we screened cell lysates generated under similar growth conditions for the presence of peptidases. This screen identified three mono aminopeptidase-type activities (active against K-pNA, F-pNA and P-pNA), as well as chymotrypsin-type (Suc-AAPF-pNA) and keratinase-type (Suc-AAA-pNA) activities. Purification and characterization of the predominant intracellular activity produced by T. emersonii, which was determined to be a proline aminopeptidase (TePAP), was successfully achieved.

The inhibition profile of purified TePAP indicated that histidine-alkylating and thiol-modifying reagents inhibit P-pNA hydrolysis, suggesting the involvement of essential, though not necessarily catalytic, Cys and His residues. However, sequence analysis identified the enzyme as a serine peptidase, complete with a conserved catalytic triad (Fig. 3). The unusual inhibitor profile of all PAPs regarding their sensitivity to thiol-modifying reagents (Supplementary Table S3) and resistance to serine peptidase inhibitors has complicated their classification. Earlier studies indicated that inhibition of Serratia marcescens PAP (SmPAP) by PCMB was the result of modification of cysteine residues located in or around the P₁ pocket (Ito et al., 2000). It should be noted, however, that no cysteine was found to be conserved in all PAPs, therefore further work is required to elucidate the cysteine peptidase-like inhibition patterns of these enzymes. The discrepancy observed between the near total inhibition of TePAP by PCMB but only partial inhibition by NEM has also been observed with other bacterial PAPs (Kitazono et al., 1996). This is consistent with PCMB being more reactive than NEM while also being capable of alkylating amines (Morty et al., 2005).

PAPs are not obligate proline aminopeptidases, as many members identified to date are capable of cleaving residues that consist of structural elements of the pyrrolidine ring of proline, such as alanine and sarcosine. In fact, some PAPs are capable of cleaving such substrates at higher rates relative to Pro–X bonds (Inoue et al., 2003). TePAP, however shows 50-fold lower activity against A-pNA relative to P-pNA (Table 1). The residues responsible for catalysis and hydrophobic pocket formation are indicated in Fig. 3(b). Some conservation is observed across all kingdoms, and it is possible that subtle differences in the micro-environment of this pocket account for the varying secondary cleavage preference noted for individual PAPs.

It is well established that the presence of extracellular inducers can affect expression levels of fungal hydrolases via specific pathways, such as cellulase induction by cellulose (Doi, 2008). The production and regulation of secreted peptidases in fungi are strongly influenced by culture conditions, although conditions for induction and/or repression appear to be enzyme- and species-specific (Polaina & MacCabe, 2007). Much less information exists regarding the regulation of intracellular fungal peptidases. The results presented here establish that TePAP is regulated at the level of transcription by environmental signals. In contrast, earlier studies of intracellular aminopeptidases from Aspergillus have indicated that expression is constitutive (Basten et al., 2001, 2003, 2005). This study definitively shows induction of TePAP when the peptide hydrolysate from casein (a protein rich in proline) is present in growth medium supplemented with a favoured carbon source (glucose). Peptides from a similar casein hydrolysate (peptone) have been shown to induce lipase production in the yeast Yarrowia lipolytica, and this induction has been attributed to the presence of specific peptides (Fickers et al., 2004). It remains possible that a similar induction pathway and therefore peptidase inducer exists for fungal peptidases like TePAP. It is interesting to note that peptides larger than six amino acids cannot be transported across the membrane of yeast (Milewski et al., 2001), implying that extensive pre-hydrolysis of proteins/peptides present in the microbial environment is required for efficient uptake. This concept supports our previous studies of secreted pepsin- and glutamic-type peptidases from T. emersonii. TGP1, a glutamic peptidase, readily cleaves Phe–Pro bonds as well as a broad range of non-proline-containing peptide bonds (O'Donoghue et al., 2008), while PEP1 (pepsin-type enzyme) cleaves Phe–Pro, Lys–Pro and Arg–Pro bonds (results not shown). The combined action of the two enzymes would generate multiple short peptides containing N-terminal proline residues for uptake. Further evidence to support this concerted digestion was derived by comparison of the expression patterns of TGP1 and TePAP. The highest expression level of tgp1 occurs after 24 h growth on glucose/casitone medium and is largely downregulated to basal levels by 48 h. However, under identical conditions, Tepap is only induced after 48 h growth, which correlates with the likely timeframe required for TGP1 to reduce extracellular protein to peptides of a suitable size for intracellular uptake. Furthermore, there is the possibility that other as yet undiscovered peptidase(s) in addition to TGP1 also provide suitable peptides for subsequent uptake and hydrolysis by PAP.

Previously, PAPs have been broadly classified into two groups on the basis of their existence as monomeric or multimeric proteins (Polaina & MacCabe, 2007) and also tentatively by substrate specificity (Yoshimoto et al., 1999). This study definitively shows that PAPs occupy at least two subfamilies that correlate well with multimerization. The 3D structures of only two PAPs have been solved (both of which are functional as monomers), from the Gram-negative bacteria Xanthomonas campestris (XcPIP; Medrano et al., 1998) and Serratia marcescens (Yoshimoto et al., 1999). These proteins are folded into two contiguous domains (Fig. 3): a central domain consisting of a β-sheet (strands β1–8) flanked by six helices (αA–F) that forms a canonical α/β hydrolase fold; and a cap domain located between β6 and αD that consists entirely of helices (αD1–6). In general, α/β hydrolase enzymes share similar topological features without extensive sequence homology, and large insertions are common (Ollis et al., 1992). Indeed, multimeric PAP sequences share low sequence identity and are ∼120 amino acids larger than their monomeric counterparts. Secondary structure analysis predicted TePAP to be composed of an α/β hydrolase fold similar to XcPIP with additional helices in the cap domain. This larger cap domain is a common feature of all PAPs in the S33.008 subfamily and therefore may play a role in multimerization.

The existence of PAPs in vastly diverse organisms and unrelated phyla (e.g. actinomycetes and proteobacteria) suggests that these unusual enzymes carry out conserved roles central to biological processes in these organisms. The presence of PAP activity may confer an adaptive advantage, as competing micro-organisms lacking this enzyme (such as Saccharomyces cerevisiae) would be unable to completely degrade high-proline-content peptides. Proline-rich proteins are abundant in nature and include plant cell wall proteins and glutens. However, the significance of proline liberation and its functions within this distinct set of micro-organisms remains to be elucidated. Bacterial PAPs are found in both S33.001 and S33.008 subfamilies with some actinomycetes and proteobacteria in opposing clades. This suggests that a subset of bacterial PAPs are more closely related to their eukaryotic counterparts than to other bacterial PAPs.

The reported role of PAPs in bacteria as virulence factors (Felipe et al., 2005) suggests that invasive organisms use this peptidase to degrade proline-rich host proteins, such as collagen in animals. Interestingly, phylogenetic analysis suggests two subclades within the S33.001 family, one of which consists of PAPs from pathogenic species that include X. campestris, S. marcescens and N. gonorrhoeae, while the other consists predominantly of non-pathogenic organisms. This subgrouping is not apparent in S33.008, as PAPs have only been characterized from non-pathogenic species.

In conclusion, TePAP is the second PAP to be characterized and cloned from the fungal kingdom. Comparative genomic analysis described here predicts the widespread occurrence of PAPs in many fungal species (Supplementary Table S4). The presence of additional sequence motifs (αM1–4), identified in this study, indicates that all eukaryotic PAPs exist as multimers. Although biochemical studies with TePAP show many similarities to PAPs from other organisms, we have identified two novel features of this peptidase. Firstly, unlike other fungal aminopeptidases, TePAP is not constitutively expressed. Possessing an inducible PAP may enable T. emersonii to adapt to and thrive on a greater diversity of nutrient sources. Secondly, this study reveals a relationship between extracellular protein degradation and intracellular induction of peptidases. The correlation between the induction and specificity of both TGP1 and TePAP suggests the existence of a pathway that regulates concerted peptidase action.

This work was supported in part by Enterprise Ireland Research Innovation Fund, Higher Education Authority PRTLI and Department of Agriculture, Food and Fisheries awards to M. G. T., and in part by National Institutes of Health Grant AI067423 to C. S. C. C. S. M was a recipient of a National University of Ireland Galway research scholarship and A. J. O'D received funding awards from the Irish Research Council for Science, Engineering, and Technology, Enterprise Ireland, County Cork Vocational Education Committee and an education abroad program fellowship from the National University of Ireland, Galway.

Edited by: M. Tien

Footnotes

^,†, Anthony J. O'Donoghue^1,2 †These authors contributed equally to this work.

The GenBank/EMBL/DDBJ accession number for the prolyl aminopeptidase sequence of Talaromyces emersonii is AF439997.

Four supplementary tables, presenting the effects of culture medium composition on intracellular peptidase activity in T. emersonii, the purification of PAP from T. emersonii, biochemically characterized PAP enzymes, and the identification of PAPs in fungi, with supplementary references, are available with the online version of this paper.