Cloning and functional expression of dipeptidyl peptidase IV from the ruminal bacterium Prevotella albensis M384T

Nicola D. Walker{dagger}, Neil R. McEwan and R. John Wallace

Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK

Correspondence
Nicola D. Walker
Nicola.Walker{at}agresearch.co.nz


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ruminal bacteria of the genus Prevotella play a crucial role in peptide breakdown in the rumen, a component of protein catabolism that leads to the inefficient use of dietary protein by ruminant animals. This is the first report of the cloning of a peptidase gene from a ruminal bacterium. Part of the dipeptidyl peptidase type IV (DPP-IV) gene from Prevotella albensis M384T was cloned using degenerate primers designed from conserved regions found within other known DPP-IV sequences. Flanking regions were determined by genomic walking. The DPP-IV gene was expressed in Escherichia coli. The cloned enzyme required a free N terminus and catalysed the removal of X-Pro dipeptide from proline-containing oligopeptides, where proline was the second residue from the N terminus. It was inhibited by serine protease inhibitors and the substrate analogue for mammalian DPP-IV, diprotin A. The properties of the cloned enzyme were similar to those of the native form in P. albensis and, in general, DPP-IVs from other organisms. The enzyme contained a conserved motif which is associated with the S9 class of prolyl oligopeptidases. The DPP-IV gene appeared not to be part of a contiguous operon. Regions with similarity to other putative promoters of Prevotella spp. were also identified. Construction of a phylogenetic tree demonstrated that the DPP-IV of P. albensis clusters with other DPP-IVs found in bacteria of the CytophagaFlexibacterBacteroidaceae (CFB) phylum, which are more closely related to eukaryotic DPP-IVs than the DPP-IV-like enzyme (PepX) of the lactic acid bacteria.


Abbreviations: DPP, dipeptidyl peptidase; MNA, 4-methoxynaphthylamide; pNA, p-nitroanilide

The GenBank accession number for the Prevotella albensis dipeptidyl peptidase type IV (DPP-IV) sequence reported in this article is AJ310187.

{dagger}Present address: AgResearch Ltd, Grasslands, Private Bag 11008, Palmerston North, New Zealand.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
One of the main goals of the ruminant nutritionist is to decrease excessive protein breakdown and ammonia production by the rumen microbial population and, as a result, to increase nitrogen efficiency in the ruminant animal (Leng & Nolan, 1984). Gram-negative bacteria from the genus Prevotella (formerly classified as Bacteroides) play a significant role in the breakdown of peptides in the rumen (Bladen et al., 1961; Russell, 1983; Depardon et al., 1996). These bacteria are predominant members of the rumen microbial population (Russell, 1983; Van Gylswyk, 1990; Wood et al., 1998) and are the only ruminal micro-organisms that have been identified to possess high dipeptidyl peptidase (DPP) activities (Wallace & McKain, 1989, 1991; McKain et al., 1992). Because this type of activity is the main mechanism by which oligopeptides are broken down in ruminal digesta (Wallace & McKain, 1989), it is likely that if either the population of these ruminal bacteria or their peptidase activities could be decreased, this could lead to an increase in the amount of nitrogen that leaves the rumen and enters the small intestine. This, in turn, may lead to a positive effect on the overall nutritional efficiency of the ruminant animal.

Four species of ruminal Prevotella have been described, differentiated on the basis of phenotypic and genetic diversity (Avgustin et al., 1994, 1997), but molecular phylogenetic analysis would suggest that many more species are present in the rumen (Wood et al., 1998). Prevotella (Pre.) albensis is one of the recognized subgroups of Prevotella found in the rumen; its type strain is M384T (Avgustin et al., 1997). Ion-exchange chromatography has revealed that this organism possesses at least four different DPPs, distinguished by their substrate and inhibitor specificity (Wallace et al., 1997). One of these peptidases displayed similar characteristics to mammalian DPP-IV: it hydrolysed recognized test substrates for DPP-IV and was inhibited by serine protease inhibitors. DPP-IV appears to have a specialized role in hydrolysing proline-containing peptides, with a wide distribution in both prokaryotes and eukaryotes (McDonald & Barrett, 1986; Misumi & Ikehara, 1998). The aim of this work was to clone the gene from Pre. albensis encoding DPP-IV and to characterize its properties both biochemically and genetically. This is the first report of the cloning and functional expression of a DPP from a ruminal bacterium.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Organisms, growth media and DNA extraction.
Pre. albensis M384T was isolated from the rumen of a sheep (Wallace & Brammall, 1985) and is maintained in the culture collection of the Rowett Research Institute. Pre. albensis M384T was grown overnight at 39 °C in the liquid form of M2 medium (Hobson, 1969), a ruminal-fluid-containing general purpose medium used for the growth of ruminal bacteria. DNA was extracted from the overnight culture using Qiagen 100-G genomic tips following the manufacturer's guidelines.

Escherichia coli TOP10 (Invitrogen) and its derivative clones were grown in LB broth containing 50 µg kanamycin ml-1 at 37 °C. Clone DPP-IV-2B contained the complete dpp-IV gene and its flanking regions and the control clone contained the 16S rRNA gene from Pre. albensis. Plasmid DNA was isolated using Wizard SV plasmid preparation columns (Promega).

PCR amplification of genomic DNA with degenerate primers based on regions of amino acid identity and cloning of PCR products.
Amino acid sequences of DPP-IVs from Porphyromonas (Por.) gingivalis and Chryseobacterium (formerly Flavobacterium) meningosepticum were aligned and two conserved regions of homology, DWVYEEE (located approximately 190 aa from the N terminus) and GWSYGG (located approximately 130 aa from the C terminus), were identified. These regions were approximately 1200 bp apart. Degenerate primers were designed by reverse translation: DPP-IV Sen1 and DPP-IV Rev1 (Table 1). Oligonucleotides were obtained from Cruachem. Thirty-six cycles of DNA amplification were performed using a denaturation temperature of 94 °C for 40 s, an annealing temperature of 50 °C for 2 min and an amplification temperature of 72 °C for 3 min. A final extension step of 72 °C for 7 min was included. The resulting amplification product was checked by electrophoresis prior to cloning and was approximately 1200 bp in size, which was in accordance with the expected size. The PCR product was purified using Wizard resin (Promega) and cloned into TOPO TA vector following the manufacturer's guidelines (Invitrogen).


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Table 1. Primers used for the amplification of the dpp-IV gene from Pre. albensis

 
Sequencing of DNA.
Plasmid DNA was sequenced in both directions on an ABI Prism 377 DNA Sequencer, using an ABI Prism BigDye terminator sequencing ready reaction kit. Due to the clone being longer than the limits of accuracy for the DNA sequencer, internal primers were used to sequence the complete clone (Table 1) and to sequence DNA by genomic walking. All sequences were translated using the Protein Translation facility at Services sur le Web d'Infobiogen (http://www.infobiogen.fr/services/menuserv.html). Similarity to putative ORFs was established using BLASTP searches (http://www.ncbi.nlm.nih.gov/BLAST/) using BLOSUM62 as the matrix.

Specific primers (DPP-IV N-term and DPP-IV C-term) were designed on the flanking regions of the dpp-IV gene (Table 1) and used to amplify a sample of genomic DNA to obtain a segment of DNA that contained the complete gene. Thirty-six cycles of DNA amplification were performed using a denaturation temperature of 94 °C for 1 min, an annealing temperature of 50 °C for 2 min and an amplification temperature of 72 °C for 5 min. A final extension step of 72 °C for 7 min was included. The resulting PCR product was cloned into TA vector as before. The size of the inserts was tested in 10 clones and the N- and C-terminal regions of the clones were sequenced to check the orientation and nature of the insert. Clones which possessed inserts of the correct size were screened for DPP-IV activity (see below) and one, DPP-IV-2B, was selected for further study.

Phylogenetic analysis and Kyte–Doolittle plot.
DPP-IV and related sequences were aligned via the internet using CLUSTAL W (Thompson et al., 1994) with the ‘aln’ option as the preferred output option. A phylogenetic tree was constructed by selecting the PHYLIP output option from CLUSTAL W. The output was analysed using programs from the PHYLIP package, version 3.57c (Felsenstein, 1989). Programs used from this package were SEQBOOT, PROTPARS and CONSENSE. The final treefile produced by the CONSENSE program was viewed using TREEVIEW (Page, 1996). A hydrophilicity profile was determined via the internet using a Kyte–Doolittle plot (Kyte & Doolittle, 1982) (http://bioinformatics.weizmann.ac.il/hydroph/plotfft_hydroph.html). The N-terminal 50 aa sequence was analysed for the presence of a putative signal peptide using the SignalP predictor service (http://www.cbs.dtu.dk/services/SignalP) (Nielsen et al., 1997).

Preparation of cell-free extracts.
A fresh overnight culture of E. coli clone DPP-IV-2B in LB medium containing 50 µg kanamycin ml-1 was harvested by centrifugation (27 500 g, 4 °C, 15 min), washed twice in 25 mM potassium phosphate buffer and resuspended in 5 ml of the same buffer. The cells were then sonicated at 30 µm (Soniprep 150; http://www.reallabware.com), for a total sonication time of 10 min on ice, in 30 s bursts with 30 s cooling intervals. Cell debris and intact cells were removed by centrifugation (15 000 g, 4 °C, 15 min) and the supernatant was retained on ice as the crude extract. Crude extracts were prepared in the same way for the control E. coli TOP10 strain which contained a 16S rRNA gene insert from Pre. albensis and for an overnight culture of Pre. albensis grown in M2 medium.

Purification of cloned DPP-IV.
Aliquots (0·5 ml) of crude extract from the control and DPP-IV-2B E. coli clones were applied to a Poros HQ anion exchange column (Perseptive Biosystems) and eluted with 20 mM Tris/HCl, pH 8·0, at 3 ml min-1 using a salt gradient of 0–1·0 M NaCl. Absorbance (at 280 nm) was monitored continuously. Fractions were tested for their peptidase activities. Several runs were performed and active fractions were pooled. These were then used to characterize the cloned enzyme.

Gel filtration was used to assess the molecular mass of the native enzyme. Three millilitres of active fraction were applied to a 100x1·0 cm column of Sephacryl-300 HR (Amersham Pharmacia Biotech). The column was eluted at 0·25 ml min-1 with 50 mM potassium phosphate buffer, pH 7·0.

Measurement of peptidase activities, effect of inhibitors and protein determination.
Peptidase activities were measured in crude extracts and purified enzyme using chromogenic and fluorogenic synthetic peptides (Wallace et al., 1997) or reverse phase HPLC analysis (Wallace & McKain, 1989). Acetylation of GlyProGly2 was performed by dissolving 20 mg peptide in 1 ml distilled water and chilling this solution on ice for 30 min. Acetic anhydride (200 µl) was added and the mixture was stirred for 1 h. It was then frozen at -70 °C and freeze-dried. The acetylated peptide was then reconstituted in distilled water. The degree of N-terminal modification was determined using ninhydrin (Moore & Stein, 1954) and demonstrated that the peptide was completely acetylated. The effect of inhibitors on peptidase activity was determined using purified enzyme by methods used previously (Wallace et al., 1997). The protein content of the cell extract was measured using the Folin reagent following alkaline digestion (Herbert et al., 1971) with bovine serum albumin as standard.

Determination of optimum pH, optimum temperature, temperature stability and Km of DPP-IV.
The pH and temperature activity profiles were determined using crude extract from the clone DPP-IV-2B, by the rate of hydrolysis of GlyPro-p-nitroanilide (GlyPro-pNA). The test buffers were: 0·1 M potassium citrate/phosphate buffer pH 2·6, 3·4, 4·2, 5·0 and 5·8; 0·2 M potassium phosphate buffer pH 6·2, 6·6, 7·0, 7·4 and 7·8; and 0·2 M Tris/HCl buffer pH 7·2, 8·0, 8·8 and 9·0. The optimum temperature of DPP-IV was determined from initial reaction rates at temperatures between 22 and 70 °C. Temperature stability was determined by incubating aliquots of the sample at 22, 30, 37, 40, 42, 45, 49, 55, 60, 63, 65 or 70 °C for 1 h before being analysed for enzyme activity.

To determine the Michaelis–Menten constant, Km, the activity of the crude extract against GlyPro-pNA was determined as before, except that the concentration of GlyPro-pNA in the incubation mixture was adjusted to 0·01–8·0 mM. The results were plotted by linear regression on a double-reciprocal plot of 1/V against 1/S where the intercept on the horizontal axis is -1/Km.

Influence of medium composition on DPP-IV activity.
A defined minimal medium (DM) was used to determine the influence of peptides and amino acids on DPP-IV activity. DM was based on the basal medium of Pittman & Bryant (1964), with added 0·1 % ammonium chloride and 0·0045 % methionine. Trypticase (1 %, w/v) or casein acid hydrolysate (1·55 %, w/v) was added to DM to determine the influence of peptidase substrates and products on DPP-IV activity. Pre. albensis M384T was grown to mid-exponential phase, then the crude extract was prepared as described for clone DPP-IV-2B. Peptidase activity was measured using GlyPro-pNA.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Amplification of genomic DNA using degenerate primers
A PCR product of the correct size, by analogy with other deposited DPP-IV sequences, of approximately 1200 bp, was obtained using degenerate primers (Table 1). Further sequence information was obtained by genomic walking and the complete gene, including the flanking regions, was obtained. The sequence was deposited in GenBank under accession number AJ310187. Sequence analysis revealed one ORF of 2193 bp. Putative promoter sites TATCTTTG and CAGAT were identified at positions -9 and -77, respectively, from the translation initiation start site by comparison with the known consensus motif obtained for promoter regions in Por. gingivalis and Bacteroides fragilis, which are closely related to Pre. albensis. Stop codons were found in all six reading frames in the flanking upstream and downstream regions of the gene, indicating that it was not part of a contiguous operon. In addition, a putative rho-independent terminator loop was identified 34 nt downstream of the stop codon.

The deduced sequence of 730 aa (Fig. 1) contained the characteristic catalytic domain GWYSGG found in all known DPP-IVs and the conserved region DWVYEEE which was used to design the other degenerate primer. The putative DPP-IV had a calculated molecular mass of 83 kDa and a theoretical pI of 7·56. The calculated G+C content of this sequence was 39 mol%. A Kyte–Doolittle hydropathy plot of the sequence was constructed (not shown), which indicated that the protein was hydrophilic in nature apart from the N-terminal region. Further SignalP analysis of the N-terminal sequence confirmed this to be a putative signal sequence, with a predicted cleavage site between residues 19 and 20.



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Fig. 1. CLUSTAL W alignment of the amino acid sequences of DPP-IVs from eukaryotes and bacteria of the CFB phylum. Conserved amino acid residues are highlighted. The amino acids that comprise the catalytic triad are marked with an asterisk. The amino acids that mark the boundaries of exons 2, 3 and 10 within the mouse sequence are marked with arrows.

 
The similarity of the putative DPP-IV with other known proteins was determined using BLASTP and compared with other known proteins in the databases. Considerable similarity was observed between the Pre. albensis DPP-IV sequence and that of Por. gingivalis DPP-IV (42 % identity), C. meningosepticum DPP-IV (39 %) and Schizosaccharomyces pombe (30 %); the mammalian DPP-IVs of rat (28 %), mouse (27 %) and human (26 %) origin also showed significant homology. The DPP-IV-like enzyme PepX of the lactic acid bacteria did not show any significant similarity (data not shown). CLUSTAL W alignment of the DPP-IV sequences of Pre. albensis, Por. gingivalis, C. meningosepticum and the eukaryotic DPP-IVs (Fig. 1) allowed the identification of the catalytic triad Ser-600, Asp-674 and His-706, and demonstrated that even between these very diverse organisms regions of homology were conserved. Comparison of the CLUSTAL W alignment of the sequences from Pre. albensis, Por. gingivalis and C. meningosepticum relative to the genomic sequence from mouse (GenBank accession nos U12599U12620) revealed that the two major deleted areas of the bacterial sequences relative to the mouse corresponded to (i) the 3' terminus of exon 2 and the 5' terminus of exon 3 and (ii) the 3' terminal region of exon 10 (Fig. 1). A phylogenetic tree was constructed (Fig. 2) which demonstrated the relatedness of the DPP-IV of Pre. albensis with other prokaryotic DPP-IVs, PepX of the lactic acid bacteria and the eukaryotic DPP-IVs.



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Fig. 2. Phylogenetic tree showing the relationships of DPP-IVs from eukaryotes and bacteria of the CFB phylum and PepX of the lactic acid bacteria. See Methods for details of tree construction. The bootstrap values denote the percentage of times a particular branch appears within the datasets used to derive the tree.

 
Purification and properties of cloned DPP-IV
Clone DPP-IV-2B was found to contain the N- and C-terminal regions of DPP-IV in the correct orientation as determined by sequence analysis. DPP activities against the chromogenic and fluorogenic test substrates for DPP-I to DPP-IV were measured and compared with a control clone which contained a 16S rRNA gene insert. Any peptidase activity observed in clone DPP-IV-2B was due to expression from the clone's own promoter. Crude extract from the control clone had an activity of 97 and 14 nmol min-1 (mg protein)-1 against Ala2-pNA and LysAla-4-methoxynaphthylamide (LysAla-MNA), activities of <5 nmol min-1 (mg protein)-1 against GlyArg-pNA, ArgArg-MNA, GlyArg-MNA, LeuVal-MNA and Leu-MNA, respectively, and undetectable [<1 nmol min-1 (mg protein)-1] activity against GlyPro-pNA and GlyPro-MNA. Clone DPP-IV-2B had a specific activity of 113 nmol min-1 (mg protein)-1 against GlyPro-pNA and 16 nmol min-1 (mg protein)-1 against GlyPro-MNA, but was otherwise identical in its activities to the control clone.

Purification of DPP-IV was carried out by anion-exchange chromatography. The DPP-IV activity was separated from the background aminopeptidase activities of E. coli, and much of the protein was also removed (Fig. 3). The fraction that contained the highest DPP-IV activity also had activity against LysAla-MNA. Active DPP-IV fractions were pooled. Only one band, of 80 kDa, was detected on an SDS-polyacrylamide gel stained with Coomassie blue and the preparation appeared to be homogeneous. Twenty-two synthetic peptides were tested for hydrolysis by the pooled fractions. Only six were hydrolysed. GlyPro-pNA, LysPro-MNA and GlyPro-MNA were hydrolysed most rapidly [900, 377 and 167 nmol min-1 (mg protein)-1, respectively], while the rates of hydrolysis of ValAla-pNA, LysAla-MNA and Ala2-pNA were lower [52, 44 and 16 nmol min-1 (mg protein)-1, respectively]. p-Nitroanilides of Ala, Arg, Asp, Glu, Gly, Leu, Lys, Met, Pro, Tyr, Val and GlyArg were not hydrolysed, nor were MNA esters of Leu, ArgArg, GlyArg or LeuVal. Thus, the DPP-IV-like enzyme did not break down aminopeptidase substrates or the test substrates for DPP-I (GlyArg-MNA) and DPP-III (ArgArg-MNA).



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Fig. 3. Anion-exchange chromatography of peptidase activities of crude cell extracts from a control clone (a) and cloned DPP-IV (b). Closed symbols represent cloned DPP-IV, open symbols represent the control clone. Activity against GlyPro-MNA ({blacklozenge}, {lozenge}), LysAla-MNA ({blacksquare}, {square}) and Leu-MNA ({blacktriangleup}, {triangleup}) was determined fluorometrically by determining the concentration of MNA released after 30 min at 39 °C. Protein content was measured by A280 ({circledast}). x, NaCl gradient.

 
The activity of the purified recombinant enzyme against di-, tri- and tetrapeptides that contained either Pro or Ala as the second residue from the N terminus was also measured. The dipeptides Ala2 and GlyPro were not hydrolysed, nor was Ala3. However, GlyProAla, GlyProGly2 and Ala4 were broken down at rates of 95, 90 and 17 nmol min-1 (mg protein)-1, respectively, by the removal of a dipeptide from the N terminus. Acetylation of the N terminus of GlyProGly2 protected it completely from breakdown.

The effect of different inhibitors on activity against GlyPro-MNA and LysAla-MNA was measured (Table 2). Only diprotin A, PMSF and dichloroisocoumarin inhibited DPP-IV activity.


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Table 2. Effect of inhibitors on peptidase activity of cloned DPP-IV

Results are the mean of triplicate incubations. The concentration of inhibitor shown is the final concentration in the incubation mixture. The function of the inhibitors is described by Benyon & Bond (1989).

 
The cloned DPP-IV had a pH optimum of 7·8 and a temperature optimum of 39 °C, exactly the same as DPP-IV activity in sonicated extract of Pre. albensis. The apparent molecular mass of the native enzyme in gel filtration was around 240 000 Da. The enzyme was stable up to 60 °C, when 1 h incubation destroyed all activity. The apparent Km of the cloned DPP-IV, with GlyPro-pNA as substrate, was 0·22 mM.

Influence of medium composition on DPP-IV activity in Pre. albensis
DPP-IV activity in defined medium, containing no added amino acids except methionine, was 12·0 (SD 1·5) nmol min-1 (mg protein)-1. In defined minimal medium supplemented with trypticase, activity increased to 21·0 (SD 3·8) nmol min-1 (mg protein)-1. Cells grown in medium containing added amino acids gave activity of 26·1 (SD 3·8) nmol min-1 (mg protein)-1.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
DPP-IV is the most highly conserved of the DPPs and is found in a wide range of different organisms, including prokaryotes and eukaryotes (Misumi & Ikehara, 1998). This conservation aided the identification of regions within this enzyme for the design of degenerate primers for the amplification of genomic DNA from Pre. albensis. The results showed that DPP-IV from Pre. albensis is similar to enzymes that belong to the S9 class of prolyl oligopeptidases (Rawlings et al., 1991). Members of this class of enzymes occur in eukaryotes and prokaryotes and are believed to be evolutionarily related (Rawlings, 1998; Fig. 1). The DPP-IV-like enzyme PepX of the lactic acid bacteria showed no amino acid sequence similarity to the DPP-IV of Pre. albensis. Therefore, the DPP-IVs of organisms that are members of the CytophagaFlexibacterBacteroidaceae (CFB) phylum (Ramsak et al., 2000), including Prevotella, Porphyromonas and Chryseobacterium, appear to be more similar to the eukaryotic DPP-IVs than to other prokaryotic DPP-IV-like enzymes, such as PepX. The S9 class also includes several proteases and endopeptidases that recognize and hydrolyse proline-containing peptides and proteins. The motif GXSXGG has been identified in all the members of the S9 class, suggesting that it is important in forming the catalytic site for the cleavage of the peptide bond between the proline and the adjacent amino acid residue. This motif also contains the serine residue that forms part of the catalytic triad associated with these serine proteases, in its centre (David et al., 1993).

A hydropathy plot identified an N-terminal region that was hydrophobic in nature, suggesting that it was associated with the cell membrane, and sequence analysis revealed a likely signal sequence. Wallace et al. (1997) found that DPP-IV was solubilized by sonication of Pre. albensis, but the enzyme may have been displaced from the membrane or from the periplasm. Other DPP-IVs are membrane-bound (Kiyama et al., 1998; Misumi & Ikehara, 1998), so the same may be true for the Pre. albensis enzyme. A membrane location may place the enzyme in close proximity to the peptide transporters that translocate the peptide across the membrane, thus placing the enzyme in an ideal location to hydrolyse its substrate.

The apparent molecular mass of the native enzyme was around 240 000 Da, in comparison with a molecular mass of 83 000 Da derived from the amino acid sequence. The cause of this difference is not clear. The active enzyme could comprise three subunits, or some association with membranous material might have altered the gel permeation properties of the native enzyme. SDS-PAGE of the purified extract indicated that the preparation was homogeneous and the purified enzyme had a molecular mass of approximately 80 kDa, which is in keeping with the theoretical mass obtained from the sequence analysis.

The dpp-IV gene of Pre. albensis is similar to the majority of peptidase genes, which do not exist as an operon, are monocistronic and are generally transcribed from a {sigma}70 promoter (Gonzales & Robert-Baudouy, 1996). Putative promoter regions were identified within the upstream region of dpp-IV (CAGAT and TATCTTTG), similar to those previously identified in Por. gingivalis (CAGAT and TATAAT; Jackson et al., 2000), as was the suggested promoter sequence for the Bacteroidaceae, including B. fragilis (TANNTTTG; Bayley & Smith, 1998). These show distinct differences from the consensus {sigma}70 -35 and -10 promoter sequence of E. coli (TTGACA and TATAAT; Hawley & McClure, 1983), not only in terms of sequence but also in terms of nucleotide spacing between the transcription start point and the individual promoter regions. There is not the optimal 17 nt spacing observed between the -10 and -35 promoter regions, and in many instances transcription initiation occurs well upstream from the translation start site.

DPP-IV activity was increased by the presence of peptides or even free amino acids in the growth medium. Thus, peptides are apparently not required to induce DPP-IV activity. This is consistent with the suggestion that peptidase activity in Pre. albensis is co-ordinately regulated along with other enzymes involved in nitrogen metabolism (Walker, 2002). The identification of a regulatory element consensus motif would further substantiate these findings. It may also lead to the identification of other genes involved in nitrogen metabolism.

Like the mammalian DPP-IV, the Pre. albensis enzyme had a preference for a proline residue at the penultimate position and hydrolysed the bond on the carboxyl side of the proline (Misumi & Ikehara, 1998). The group beyond the penultimate amino acid affected the rate of hydrolysis, in that GlyPro-pNA was more rapidly hydrolysed by DPP-IV than GlyPro-MNA. This may be due to differences in the steric or hydrophobic nature of the chromogenic/fluorogenic group. The proline as penultimate N-terminal amino acid could also be substituted by alanine, although the rate of hydrolysis was significantly decreased. This activity was also observed in the other DPP-IVs and in PepX of the lactic acid bacteria (Chich, 1998; Misumi & Ikehara, 1998). The ability to recognize and hydrolyse more than one type of substrate indicates a degree of overlapping specificity in the DPP-IV of Pre. albensis. Overlapping specificity is also found in the peptidases of other bacteria (Lazdunski, 1989; Mierau et al., 1996; Yen et al., 1980). Studies in which peptidase-deficient mutants of Prevotella bryantii were isolated (Madeira et al., 1997) demonstrated that there must be a degree of overlapping specificity between different peptidases in this organism because peptidase activity could not be completely abolished in mutants that were deficient for DPP-I.

The DPP-IV from Pre. albensis was inhibited by dichloroisocoumarin and PMSF, recognized serine protease inhibitors, and also by diprotin A (Ile-Pro-Ile), which is a substrate-analogue inhibitor of DPP-IVs. These inhibitors have already been shown to inhibit DPP-IV activity in crude cell extracts of Pre. albensis (Wallace et al., 1997), once again indicating the close similarity between the cloned enzyme and DPP-IV of Pre. albensis. The optimum pH (7·8) and broad pH range over which the enzyme was active was also similar to mammalian DPP-IVs (Misumi & Ikehara, 1998), as was the optimum temperature (39 °C in comparison to 37 °C). Here, the pH and temperature optima were determined using crude extract from sonicated clone DPP-IV-2B; as the control clone with a 16S rRNA gene insert had no detectable DPP-IV activity, the results can be expected to be valid for the recombinant DPP-IV. Thus, the biochemical properties, in conjunction with the sequence data, of the first peptidase to be cloned from a ruminal bacterium indicate that it has properties very similar to DPP-IVs from closely related Gram-negative bacteria, such as Porphyromonas, and to DPP-IVs in distantly related mammalian species, despite very different biochemical conditions, environmental circumstances and, possibly, metabolic functions. It remains to be seen how the other main DPPs of Pre. albensis fit into the wider scheme of peptidase enzymes.


   ACKNOWLEDGEMENTS
 
The Rowett Research Institute receives core funding from the Scottish Executive Environment and Rural Affairs Department.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Bayley, D. P. & Smith, C. J. (1998). The unique promoters of Bacteroides fragilis. In Abstracts of the 98th General Meeting of the American Society for Microbiology 1998, abstract H-101, p. 293. Washington, DC: American Society for Microbiology.

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Chich, J.-F. (1998). X-Pro dipeptidyl peptidase. In The Handbook of Proteolytic Enzymes, pp. 403–405. Edited by A. J. Barrett, N. D. Rawlings & J. F. Woessner. London: Academic Press.

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Received 11 November 2002; accepted 26 March 2003.



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