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Department of Microbiology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain
Correspondence
Francisco I. Javier Pastor
fpastor{at}ub.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Pectin degradation requires the combined action of several enzymes, among which two main groups can be distinguished: pectinesterases, which remove methoxyl groups from pectin, and depolymerases (hydrolases and lyases) that degrade the backbone chain. Lyases cleave glycosidic bonds by 
-elimination, giving rise to unsaturated products. Among these enzymes, pectate lyases are specific for unmethylated polygalacturonate (pectate), although they can be active on pectins with a low degree of methyl esterification (Benen & Visser, 2003; Tardy et al., 1997). Pectate lyases are widely distributed among microbial plant pathogens, where they play an important role as virulence factors (Barras et al., 1994; Herron et al., 2000). They have also been found in saprophytic micro-organisms, including members of the genus Bacillus (Berensmeier et al., 2004; Hatada et al., 2000; Nasser et al., 1993) and in some thermophilic bacteria (Kluskens et al., 2003). Multiple sequence alignment of pectate lyases has been used to classify these enzymes into several subfamilies and to identify consensus sequences of some of them (Coutinho & Henrissat, 1999; Shevchik et al., 1997). The study of the three-dimensional folding of pectate lyase C from Erwinia chrysanthemi led to the finding of a novel protein structure, a parallel-right handed -helix (Yoder et al., 1993), that is found in pectate lyases of families 1, 3 and 9 (Jenkins et al., 2004).
Pectin-degrading enzymes are widely used in the food industry for improving the yield and the clarification of fruit juices (Kashyap et al., 2001). Alkaline pectinases are being introduced in the textile industry to release fibres from flax stems, as an alternative to conventional retting (Hoondal et al., 2002). In these fields, finding new enzymes has special interest to improve the efficiency of the production systems.
In a previous study, pectate lyase PelA from Paenibacillus barcinonesis was identified and characterized (Soriano et al., 2000). As a distinctive trait among known pectate lyases, the new enzyme showed high activity on both polygalacturonate and highly methylated pectins. Analysis of the PelA amino acid sequence showed that the enzyme did not display homology to pectate lyases from saprophytic micro-organisms, while high homology was found to the hypothetical protein deduced from the yvpA gene from Bacillus subtilis (Kunst et al., 1997). In this article we describe the cloning of yvpA, and show that it encodes a pectin-degrading enzyme that has been denominated PelC. The new pectate lyase shares with PelA a high activity on highly methylated pectins and defines, together with PelA, a cluster of family 3 pectate lyases from non-pathogenic micro-organisms.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and E. coli BL21(DE3) (Studier & Moffatt, 1986) were cultivated at 37 °C in LB medium and used as recipient strains for recombinant plasmids. Plasmids pJF118HE (Fürste et al., 1986) and pET28a (Studier & Moffatt, 1986) were used as cloning vectors. E. coli/pP22 has been previously described (Soriano et al., 2000). It harbours recombinant plasmid pP22 containing the pelA gene from Paenibacillus barcinonensis (Sánchez et al., 2005) cloned in pBR322.
Nucleic acid manipulation.
Isolation of plasmids, restriction digestion of DNA, ligation of DNA and transformation of E. coli were carried out by standard methods. Chromosomal DNA from B. subtilis and P. barcinonensis was prepared as previously described (Soriano et al., 2000). PCR amplification and sequencing of DNA were done according to standard methodology.
The yvpA gene was PCR amplified with Pfu (Stratagene) with oligonucleotides 5'-ATCACCCGGGAAGATCGATTC-3' and 5'-CCGGAGCTCTTGTCCATAAG-3'. These oligonucleotides are located 179 bp upstream and 141 bp downstream of the initiation and stop codons, respectively, of yvpA structural gene. The amplified fragment was ligated to plasmid pJF118HE previously digested with SmaI, and introduced by transformation into E. coli 5K. The recombinant strain containing the yvpA gene was denominated E. coli/pJYvpA.
Plasmids for overexpression of pectate lyases under the control of the T7 RNA polymerase system (Studier & Moffatt, 1986) were constructed as follows. yvpA was amplified with oligonucleotides 5'-TTGCTAGCGCTGACAAAGTGG-3'and 5'-CTAAGCTTGTTAAAATTGAGTGTTG-3'. The amplified fragment was digested with NheI and HindIII, inserted between the NheI and HindIII sites of plasmid pET28a, and transformed into E. coli BL21(DE3). The recombinant plasmid pET28a-YvpA obtained encoded PelC, devoid of its signal peptide, with an N-terminal His-tag. After removal of the His-tag, the amino acid sequence Gly-Ser-His-Met-Ala-Ser remained added to mature PelC. For expression of pelA from P. barcinonensis, a similar approach was used. The oligonucleotides used for pelA amplification were 5'-CCCATATGGCGCCAACCGTC-3' and 5'-GCAAGCTTTAATACTGTGTATTTCCG-3'. The amplified fragment was digested with NdeI and HindIII, and inserted between the corresponding sites of pET28a. The recombinant plasmid pET28a-PelA encoded a mature PelA with an N-terminal Gly-Ser-His-Met extension, after removal of the His-tag.
Purification of pectate lyases.
Purification of PelC and PelA was performed by His-tag affinity chromatography using NTA-Ni agarose resin (Qiagen) essentially as described by the pET28a supplier (Novagen). Recombinant clones E. coli/pET28a-YvpA and E. coli/pET28a-PelA were cultivated at 37 °C in 1 litre of kanamycin-supplemented LB to OD600 0·5–0·6. Then 1 mM IPTG was added and the cultures were further incubated for 150 min. Cells were collected, resuspended in 20 ml buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8·0) and lysed in a French press. Cell extracts were clarified by centrifugation and His-tagged proteins were purified from the extracts. The His-tag of purified proteins was removed using the Thrombin Cleavage Capture Kit from Novagen.
Enzyme assays.
Pectate lyase activity was assayed spectrophotometrically by measuring the formation of unsaturated products from pectic substrates at 232 nm (Collmer et al., 1988). The standard assay mixture contained 0·2 % (w/v) substrate (Sigma) in a final volume of 3 ml 50 mM glycine buffer pH 10·0, containing 0·5 mM CaCl2 for PelA activity determination or 2 mM CaCl2 for PelC activity determination. Assay mixtures were incubated for 2·5 min at 50 °C and the increase of A232 was measured. The substrates used were polygalacturonic acid and pectins of various degrees of methylation (22, 64 and 89 % esterified citrus pectin, Sigma). Apple pectin (68 % esterification, Sigma) was also tested as a substrate. One unit of enzymic activity was defined as the amount of enzyme that produces 1 µmol unsaturated product per minute under the assay conditions described. To study the effect of pH on activity, assays were performed at 65 °C (PelC) or 55 °C (PelA), replacing 50 mM sodium glycine buffer pH 10·0 by the following buffers: 50 mM sodium citrate pH 3·0–4·0, 50 mM sodium acetate pH 4·0–6·0, 50 mM sodium phosphate pH 6·0–8·0, 50 mM Tris/HCl pH 8·0–9·0, and 50 mM sodium glycine pH 9·0–12·0. The effect of temperature was studied at pH 10 in the temperature range 30–85 °C. For inhibition studies, 1 mM different ions were added to the assay mixture and activity was determined as indicated.
For stability assays, enzymes were incubated at 50 °C in buffers at pH 7·0 or 10·0 and samples were taken at regular intervals. Residual activity after thermal treatment was determined by the standard assay.
Kinetic parameters Vmax and Km were determined by fitting hyperbolic Michaelis–Menten curves with the program SigmaPlot version 8.0 (SPSS). Kinetic parameters of PelC and PelA on 22 % methyl esterified pectin and polygalacturonic acid were determined with concentrations of substrates from 0·25 to 6 mg ml–1, at 65 °C and 55 °C, respectively, in buffers at pH 10.
Analytical procedures.
SDS-PAGE was performed in 13 % (w/v) gels, essentially as described by Laemmli (1970). Samples were heated for 10 min at 45 °C in sample buffer before being applied to gels. After electrophoresis, gels were soaked in 2·5 % (w/v) Triton X-100 for 30 min, washed in 100 mM glycine buffer pH 10·0, 1·5 mM CaCl2 for 30 min, and overlaid with a 1 % (w/v) agarose gel containing 1 % (w/v) pectin in the same buffer as above. After 30 min incubation at 45 °C, gels were stained with 0·05 % (w/v) ruthenium red (Sigma) for 10 min and washed with water until pectate lyase bands became visible.
Analysis of PelC and PelA reaction products was performed by TLC on silica gel plates (Kieselgel 60 F254, Merck). The solvent used was n-butanol/water/acetic acid (5 : 3 : 2, by vol.). Oligosaccharides were detected by spraying the dried plates with a solution of 3 % (w/v) phosphomolybdic acid and 10 % (v/v) sulphuric acid in ethanol, and heating at 125 °C for 5 min. Galacturonic, digalacturonic and trigalacturonic acids (Sigma) were used as size markers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The deduced amino acid sequence of the enzyme, PelA, did not show homology to pectate lyases from saprophytic micro-organisms, but it displayed high homology to the protein deduced from yvpA gene from Bacillus subtilis, of unknown function (Kunst et al., 1997), indicating that it probably encoded a pectin-degrading enzyme. yvpA from B. subtilis was isolated by PCR and cloned in E. coli. The recombinant clone obtained, E. coli/pJYvpA, produced haloes of substrate degradation when grown on polygalacturonic acid supplemented agar plates, showing that yvpA encoded a pectinase. Cell extracts from the recombinant clone showed lyase activity on polygalacturonic acid and pectin, while hydrolase activity on these substrates was undetectable. The results suggested that yvpA encoded a pectate lyase with similar substrate specificity to PelA.
Sequence analysis
The nucleotide sequence of the cloned gene was identical to that reported for yvpA (Kunst et al., 1997). It encodes a protein of 221 amino acids with predicted molecular mass and pI of 24281·4 Da and 8·85, respectively. The deduced amino acid sequence shows an N-terminal region of 27 amino acids with the features of a typical Bacillus signal peptide (Nagarajan, 1993). The open reading frame starts with TTG as initiation codon. This start codon is frequently found in Bacillus genes and in some instances it has been correlated to translation efficiency (Vellanoweth, 1993). Seven nucleotides upstream of the start codon there is a GGGGAGGA sequence, resembling that of a ribosome-binding (Shine–Dalgarno) site. A putative promoter sequence, similar to those recognized by the 
A subunit of Bacillus RNA polymerase, showing a –35 (TTTACT) and –10 (TATGAT) region, was found upstream of the yvpA structural gene. An inverted repeat of 14 bp, that could act as a transcriptional terminator, was found in the 3' region of the structural gene.
The deduced amino acid sequence of the cloned enzyme was compared to protein sequences in the Swissprot and EMBL databases. Homology was found to pectate lyases belonging to family PL3, while no homology was found to pectate lyases of other families (Fig. 1). The highest homology was found to pectate lyase from Bacillus sp. P-2850 (accession no. Q8L0R5), the protein deduced from yvpA gene from Bacillus licheniformis (accession no. Q65EF5), pectate lyase from Bacillus sp. KSM-P15 (Hatada et al., 2000) and PelA from P. barcinonesis (Soriano et al., 2000), which showed 72, 72, 53 and 53 % identity, respectively, to the cloned enzyme. High homology was also found to open reading frames deduced from genes SCO1110 from Streptomyces coelicolor (Bentley et al., 2002) and SAV6383 from Streptomyces avermitilis (Omura et al., 2001), which showed 44 and 40 % identity to the cloned enzyme. Homology was also found to pectate lyases from phytopathogenic fungi and bacteria: Pl1 from Fusarium oxysporum (Huertas-González et al., 1999), PelI from Erwinia chrysanthemi (Shevchik et al., 1997), PelB from Fusarium solani (Guo et al., 1995) and Pel3 from Erwinia carotovora (Liu et al., 1994), which showed 43, 42, 37 and 35 % identity, respectively, to the cloned pectate lyase.
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) and a pectin lyase (Sakamoto et al., 1996), characterized to date from B. subtilis. We named the cloned enzyme PelC and propose the encoding gene to be designated pelC instead of yvpA. The amino acid sequence of PelC contains three out of the four blocks of conserved residues proposed as signatures of pectate lyases from family 3 (Shevchik et al., 1997) (Fig. 1
). Among the conserved amino acids, PelC shows an Arg residue (Arg-158) in an identical position to Arg-132 of the mature pectate lyase from Bacillus sp. KSM-P15, which is proposed to initiate proton abstraction during the -elimination catalytic mechanism (Akita et al., 2001). The three-dimensional structure of PelC was predicted by the Swiss pdb viewer program. The enzyme folds as an eight-turn right-handed parallel -helix, similarly to family 3 pectate lyases.
Substrate specificity
The cloned pectate lyase was purified by His-tag affinity chromatography, the His-tag was removed and the properties of the enzyme were studied. Analysis by SDS-PAGE, IEF and zymograms showed that PelC has an apparent molecular mass of 23 kDa (Fig. 2) and a pI of 8·6 (data not shown), in good agreement with the predicted values of 24281·4 Da and 8·55, respectively. The influence of substrate methylation on enzymic activity was tested by evaluating the activity of the purified enzyme on polygalacturonic acid and on pectins of increasing degree of methyl esterification. Release of esterified methyl groups from pectin by saponification can take place after incubation at pH 10 for longer than 10 min (Brown et al., 2001). To avoid this phenomenon, activity was determined in 2·5 min assays in which enzyme samples, substrates and buffer at pH 10 were mixed immediately before the assays. PelC exhibited activity on all substrates tested and showed maximum activity on pectin with a low degree of methylation (22 % esterified) (Fig. 3). On polygalacturonic acid, and pectins of 64 % and 89 % methyl esterification, the enzyme showed 29, 87 and 43 %, respectively, of maximum activity.
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Pectate and pectin lyases can be distinguished by the absolute Ca2+ requirement for catalysis of pectate lyases versus the lack of Ca2+ requirement by pectin lyases (Jurnak et al., 1996). Analysis of the effect of Ca2+ on PelC activity showed that the enzyme exhibited maximum activity at 2 mM CaCl2, while without Ca2+ addition no activity on polygalacturonic acid and very little activity on pectins were found (Fig. 3). The activity found on pectin without exogenous Ca2+ could result from the presence of trace amounts of contaminant Ca2+ ions in this substrate. In fact, activity was not detected when the chelator EDTA was included in the assays. The absolute requirement of Ca2+ ions for activity indicates that PelC is a pectate lyase, although, unusually, it shows higher activity on pectins than on polygalacturonic acid and shows activity on highly methylated pectins.
Comparison to PelA from P. barcinonensis
The high activity of PelC on pectins resembled that of PelA from P. barcinonensis. This enzyme had been previously characterized from crude cell extracts from the recombinant clone E. coli/pP22 (Soriano et al., 2000). To compare the two pectate lyases more accurately, PelA was purified by His-tag affinity chromatography, and its properties were analysed after removal of the His-tag (Fig. 2). Activity of purified PelA on substrates of different degree of methyl esterification was evaluated. PelA showed maximum activity on polygalacturonic acid, although very similar activity (99 % of maximum) was found on 22 % esterified pectin (Fig. 3). On highly methylated pectin (89 % esterification) the enzyme showed 39 % of maximum activity. Activity of the enzyme on all these substrates showed an absolute requirement of Ca2+, as no activity was found in the presence of EDTA. Maximum activity was found when 0·5 mM CaCl2 was added to the assays (Fig. 3).
The effect of metal ions others than Ca2+ was analysed by performing activity assays in the presence of metal ions at 1 mM concentration, without Ca2+ added. In the case of PelA, activity was undetectable or very low in all assays, indicating that none of the ions studied could replace Ca2+. However, when PelC activity was assayed in the presence of 1 mM Hg2+, similar activity to control assays was found, suggesting that Hg2+ could replace Ca2+. When the assays were performed in the simultaneous presence of Ca2+ and Hg2+, each at 1 mM, a stimulation of PelC activity (120 % of control) was found.
The effect of pH and temperature on activities of PelC and PelA was determined. The two enzymes exhibited activity only in a narrow alkaline pH range, with maximum activity at pH 10 (Fig. 2). PelC showed maximum activity at 65 °C, while the optimum temperature for PelA activity was 55 °C (Fig. 2). Stability assays showed that PelC was stable for at least 24 h at 50 °C in buffers at pH 7, whereas PelA showed only 40 % of initial activity after incubation under these conditions. When stability assays were performed at 50 °C in buffers at pH 10, PelC showed 58 % of initial activity after 24 h incubation, while PelA was completely inactivated by this treatment.
The kinetic parameters of purified pectate lyases on pectin and polygalacturonic acid were determined. PelC showed a Vmax of 256·7±23·9 units mg–1 and a Km of 0·13±0·03 mg ml–1 on 22 % esterified pectin, while on polygalacturonic acid it showed a Vmax of 59·6±14·6 units mg–1 and a Km of 0·15±0·06 mg ml–1 (means±SE, n=3). Kinetic parameters of PelA on 22 % esterified pectin were Vmax 102·4±5·8 units mg–1 and Km 0·14±0·02 mg ml–1, while on polygalacturonic acid they were Vmax 95·4±8·3 units mg–1 and Km 0·16±0·04 mg ml–1 (means±SE, n=3).
Analysis of reaction products
To determine the mode of action of PelC and PelA, the reaction products obtained from various substrates were characterized by TLC. Both enzymes produced a mixture of oligomers from polygalacturonic acid (Fig. 4). The main product released from this substrate by PelC showed a mobility corresponding to unsaturated trigalacturonic acid. Unsaturated digalacturonic acid and oligomers of higher degree of polymerization were also released by the enzyme. PelA produced a mixture of reaction products from polygalacturonic acid similar to that produced by PelC, although the main products were oligomers longer than unsaturated trigalacturonate. Analysis of the reaction products from pectin showed that PelC and PelA released a mixture of oligomers with a similar pattern to that obtained from polygalacturonic acid. In contrast to the similar action of PelC and PelA on polygalacturonic acid and pectin, the enzymes behaved differently on trigalacturonate. PelC degraded trigalacturonate to galacturonic acid and unsaturated digalacturonate, whereas PelA was not active on this substrate.
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DISCUSSION |
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-elimination. The enzyme does not show homology to the two pectinases, a pectate lyase (Nasser et al., 1993) and a pectin lyase (Sakamoto et al., 1996), characterized up to now from this species. As no hydrolases acting on pectic polymers have been described in B. subtilis, the enzyme constitutes the third pectin depolymerase characterized in this species, and has been denominated PelC. The fact that until our work only two pectinases had been identified from B. subtilis contrasts with the pectin-depolymerizing activity of this species (Chesson & Codner, 1978) and the multiplicity of pectinases described in pectin-degrading micro-organisms, which show complex sets of enzymes. In this way, at least eight pectate lyases have been cloned and characterized in Erwinia chrysanthemi (Shevchik et al., 1999b), while Aspergillus niger contains several pectin lyases, a pectate lyase and a family of polygalacturonases (Pa
eniková et al., 2000). We believe that B. subtilis, besides the three pectin-degrading enzymes characterized up to now, has several as yet unidentified pectinases and may exhibit a complex set of enzymes for pectin degradation.
One of the most notable features of PelC is its activity on both polygalacturonic acid and highly methylated pectin. Although many pectate lyases show maximum activity on polygalacturonic acid, some are most active on low methylated substrates (7–22 % esterification) (Tardy et al., 1997). The activity of these enzymes on pectin could indicate that they are pectin lyases rather than pectate lyases, although a clear difference between the two types of enzymes is the absolute Ca2+ requirement for catalysis of pectate lyases, while pectin lyases do not require Ca2+ (Jurnak et al., 1996). Our results show that B. subtilis PelC requires added Ca2+ for activity on pectic subtrates, indicating that it is a pectate lyase. The enzyme shows maximum activity on 22 % methylated pectin, similar to what has been reported for Er. chrysanthemi PelB (Tardy et al., 1997). However, in contrast to known pectate lyases, PelC shows high activity (43 % of maximum activity) on 89 % methylated pectins. This is a unique trait among pectate lyases characterized to date, which are not active or show very low activity on highly methylated pectins (Benen & Visser, 2003; Hugouvieux-Cotte-Pattat et al., 1996), although some pectate lyases of family 3, such as PelI from Er. chrysanthemi and PelB from Er. carotovora, exhibit maximum activity on 45 % and 68 % esterified pectin, respectively (Shevchik et al., 1998; Heikinheimo et al., 1995). Pectate lyase PelA from P. barcinonensis (Soriano et al., 2000), the enzyme that led to identification of PelC, resembles this enzyme in its high activity on highly esterified pectin (39 % of maximum activity on 89 % methylated pectin). This is a difference between these two enzymes (PelC and PelA) and the pectate lyases described up to now.
Sequence comparison with proteins contained in databases revealed that PelC from B. subtilis and PelA from P. barcinonensis belong to family 3 pectate lyases. Sequence alignment of enzymes of this family has identified four blocks of conserved residues, proposed as signatures of pectate lyases of family 3 (Shevchik et al., 1997). These blocks of residues are also found in pectate lyases from plant-parasitic nematodes (Huang et al., 2005). However, the two pectate lyases characterized in this work, together with pectate lyase from Bacillus sp. P-2850 (accession no. Q8LOR5) and pectate lyase from Bacillus sp. KSM-P15 (Hatada et al., 2000) do not show the fourth block of conserved residues. In the corresponding area of these four enzymes (residues 172–221 in the case of PelC) analysis with ProDom at NCBI (Servant et al., 2002) has identified domain ID:PD346936, not found in other enzymes. The four pectate lyases show high homology (more than 50 % identity in amino acid sequence), similar size (221–224 amino acids), and low content of cysteine residues (1–3) compared to the rest of the enzymes of family 3, which show a higher cysteine content [12 residues in the case of PelB from Fusarium solani (Guo et al., 1995)]. The results show that PelC from B. subtilis and PelA from P. barcinonensis, together with pectate lyase from Bacillus sp. P-2850 and pectate lyase from Bacillus KSM-P15, constitute a subgroup of homology among family 3 pectate lyases. A dendrogram of enzymes of family 3 generated with CLUSTAL W (Thompson et al., 1994) showed that these four pectate lyases cluster separately from the rest of enzymes of family 3 (data not shown), and form a subgroup of pectate lyases from saprophytic bacteria in a family where most of the enzymes belong to pathogenic micro-organisms. Analysis of the hypothetical protein deduced from the yvpA gene from B. licheniformis (accession no. Q65EF5) reveals it shares all the common traits of the enzymes of the subgroup identified and clusters with them.
As mentioned above, PelC and PelA have as a distinctive character their high activity on pectin with a high degree of methyl esterification. This property is not shared by the other enzymes of the homology subgroup. While activity results of pectate lyase from Bacillus sp. P-2850 have not been reported, pectate lyase from Bacillus sp. KSM-P15 shows a clear preference for polygalacturonic acid and very low activity on methylated pectins (Kobayashi et al., 1999). Despite their similarity, PelC and PelA show some differences regarding substrate specificity. PelC shows a clear preference for 22 % methylated pectin, whereas PelA shows similar activity on polygalacturonic acid and 22 % esterified pectin. Study of the mode of action on polymeric substrates showed that PelC and PelA have an endo mode of action, producing a mixture of degradation products. A similar mode of action has been reported for pectate lyases of family 1, such as pectate lyase A from Clostridium cellulovorans (Tamaru & Doi, 2001), family 3, such as PelI from Er. chrysanthemi (Shevchik et al., 1997), and family 9, such as PelL from Er. chrysanthemi (Lojkowska et al., 1995). In contrast, PelX and PelW from Er. chrysanthemi (Shevchik et al., 1999a, b), which belong to families 9 and 2, respectively, show an exo mode of action, releasing unsaturated digalacturonate as the only degradation product from polygalacturonic acid. PelA and PelC differed in their activity on trigalacturonate: PelA did not degrade this substrate, whereas PelC cleaved it to galacturonate and unsaturated digalacturonate. The lack of trigalacturonate degradation by PelA is similar to that reported for endopectate lyase A from Clostridium cellulovorans (Tamaru & Doi, 2001) and to the requirement for substrates longer than a trimer for effective catalysis of several endopectate lyases from Er. chrysanthemi (Roy et al., 1999). However, degradation of trigalacturonate by PelC resembles the results described for exopectate lyases PelX and PelW of Er. chrysanthemi (Shevchik et al., 1999a, b). The scarcity of reports about the use of trigalacturonate as a substrate by pectate lyases makes it difficult to understand the significance of the different behaviour of PelA and PelC on this oligomer.
In summary, we have purified and characterized two novel pectate lyases, PelC from B. subtilis and PelA from P. barcinonensis, which differ from known pectate lyases in their high activity on highly methylated pectin. The characterized enzymes, together with pectate lyase from Bacillus sp. P-2850 and pectate lyase from Bacillus sp. KSM-P15, constitute a subgroup of highly homologous enzymes in family 3. The biochemical properties of the four pectate lyases need to be studied in more detail to identify common features of this subgroup of enzymes, which to our knowledge are the only pectate lyases from non-pathogenic micro-organisms classified in family PL3.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barras, F., van Gijsegem, F. & Chatterjee, A. K. (1994). Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopathol 32, 201–234.
Benen, J. A. E. & Visser, J. (2003). Pectate and pectin lyases. In Handbook of Food Enzymology, pp. 1029–1041. Edited by J. R. Whitaker, A. Voragen & D. W. S. Wong. New York: Dekker.
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M. & 40 other authors (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147.[CrossRef][Medline]
Berensmeier, S., Singh, S. A., Meens, J. & Buchholz, K. (2004). Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase. Appl Microbiol Biotechnol 64, 560–567.[CrossRef][Medline]
Brown, I. E., Mallen, M. H., Charnock, S. J., Davies, G. J. & Black, G. W. (2001). Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module. Biochem J 355, 155–165.[CrossRef][Medline]
Chesson, A. & Codner, R. C. (1978). The maceration of vegetable tissue by a strain of Bacillus subtilis. J Appl Bacteriol 44, 347–364.
Collmer, A., Ried, J. L. & Mount, M. S. (1988). Assay methods for pectic enzymes. Methods Enzymol 161, 329–335.[CrossRef]
Coutinho, P. M. & Henrissat, B. (1999). Carbohydrate-active enzymes: an integrated database approach. In Recent Advances in Carbohydrate Bioengineering, pp. 3–12. Edited by H. J. Gilbert, G. J. Davies, B. Henrissat & B. Svensson. Cambridge: Royal Society of Chemistry.
Fürste, J. P., Pansegrau, W., Frank, R., Bloecker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986). Molecular cloning of the plasmid RP-4 primase region in a multi-host-range Tac-P expression vector. Gene 48, 119–132.[CrossRef][Medline]
Guo, W., González-Candelas, L. & Kolattukudy, P. E. (1995). Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris. J Bacteriol 177, 7070–7077.
Hatada, Y., Saito, K., Koike, K., Yoshimatsu, T., Ozawa, T., Kobayashi, T. & Ito, S. (2000). Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkalophilic strain of Bacillus. Eur J Biochem 267, 2268–2275.[Medline]
Heikinheimo, R., Flego, D., Pirhonen, M., Karlsson, M. B., Eriksson, A., Mäe, A., Köiv, V. & Palva, E. T. (1995). Characterization of a novel pectate lyase from Erwinia carotovora subsp. carotovora. Mol Plant Microbe Interact 8, 207–217.[Medline]
Herron, S. R., Benen, J. A. E., Scavetta, R. D., Visser, J. & Jurnak, F. (2000). Structure and function of pectic enzymes: virulence factors of plant pathogens. Proc Natl Acad Sci U S A 97, 8762–8769.
Hoondal, G. S., Tiwari, R. P., Tewari, R., Dahiya, N. & Beg, Q. K. (2002). Microbial alkaline pectinases and their industrial applications: a review. Appl Microbiol Biotechnol 59, 409–418.[CrossRef][Medline]
Huang, G., Dong, R., Allen, R., Davis, E. L., Baum, T. J. & Hussey, R. S. (2005). Developmental expression and molecular analysis of two Meloidogyne incognita pectate lyase genes. Int J Parasitol 35, 685–692.[CrossRef][Medline]
Huertas-González, M. D., Ruiz-Roldan, M. C., Garcia-Maceira, F. I., Roncero, M. I. & Di Pietro, A. (1999). Cloning and characterization of pl1 encoding an in planta-secreted pectate lyase of Fusarium oxysporum. Curr Genet 35, 36–40.[CrossRef][Medline]
Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W. & Reverchon, S. (1996). Regulation of pectinolysis in Erwinia chrysanthemi. Annu Rev Microbiol 50, 213–257.[CrossRef][Medline]
Jenkins, J., Shevchik, V. E., Hugouvieux-Cotte-Pattat, N. & Pickersgill, R. W. (2004). The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi. J Biol Chem 279, 9139–9145.
Jurnak, F., Kita, N., Garrett, M., Heffron, S. E., Scavetta, R., Boyd, C. & Keen, N. (1996). Functional implications of the three-dimensional structures of pectate lyases. In Pectin and Pectinases, vol. 14, pp. 295–308. Edited by J. Visser & A .G. J. Voragen. Amsterdam: Elsevier.
Kashyap, D. R., Vohra, P. K., Chopra, S. & Tewari, R. (2001). Applications of pectinases in the commercial sector: a review. Bioresource Technol 77, 215–227.[CrossRef][Medline]
Kluskens, L. D., Van Alebeek, G. J. W. M., Voragen, A. G. J., De Vos, W. M. & Van Der Oost, J. (2003). Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima. Biochem J 370, 651–659.[CrossRef][Medline]
Kobayashi, T., Koike, K., Yoshimatsu, T., Higaki, N., Suzumatsu, A., Ozawa, T., Hatada, Y. & Ito, S. (1999). Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus. Biosci Biotechnol Biochem 63, 65–72.[CrossRef][Medline]
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Liu, Y., Chatterjee, A. & Chatterjee, A. K. (1994). Nucleotide sequence and expression of a novel pectate lyase gene (pel-3) and a closely linked endopolygalacturonase gene (peh-1) of Erwinia carotovora subsp. carotovora 71. Appl Environ Microbiol 60, 2545–2552.
Lojkowska, E., Masclaux, C., Boccara, M., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1995). Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937. Mol Microbiol 16, 1183–1195.[Medline]
Nagarajan, V. (1993). Protein secretion. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, pp. 713–726. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
Nasser, W., Awadé, A. C., Reverchon, S. & Robert-Baudouy, J. (1993). Pectate lyase from Bacillus subtilis: molecular characterization of the gene, and properties of the cloned enzyme. FEBS Lett 335, 319–326.[CrossRef][Medline]
Omura, S., Ikeda, H., Ishikawa, J & 11 other authors (2001). Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci U S A 98, 12215–12220.
Paeniková, L., Benen, J. A. E., Kester, H. C. M. & Visser, J. (2000). pgaA and pgbB encode two constitutively expressed endopolygalacturonases of Aspergillus niger. Biochem J 345, 637–644.
Roy, C., Kester, H., Visser, J., Shevchik, V., Hugouvieux-Cotte-Pattat, N., Robert-Baudouy, J. & Bennen, J. (1999). Modes of action of five different endopectate lyases from Erwinia chrysanthemi 3937. J Bacteriol 181, 3705–3709.
Sakamoto, T., Kawasaki, H. & Sakai, T. (1996). Molecular cloning and nucleotide sequence of the gene encoding phosphate-inducible pectin lyase of Bacillus subtilis. FEBS Lett 398, 269–273.[CrossRef][Medline]
Sánchez, M. M., Fritze, D., Blanco, A., Spröer, C., Tindall, B. J., Schumann, P., Kroppenstedt, R. M., Diaz, P. & Pastor, F. I. J. (2005). Paenibacillus barcinonensis sp. nov., a xylanase-producing bacterium isolated from a rice field in the Ebro River delta. Int J Syst Evol Microbiol 55, 935–939.
Schols, H. A. & Voragen, A. (2003). Pectic polysaccharides. In Handbook of Food Enzymology, pp. 829–843. Edited by J. R. Whitaker, A. Voragen & D. W. S. Wong. New York: Dekker.
Servant, F., Bru, C., Carrère, S., Courcelle, E., Gouzy, J., Peyruc, D. & Kahn, D. (2002). ProDom: automated clustering of homologous domains. Brief Bioinform 3, 246–251.
Shevchik, V. E., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1997). Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family. J Bacteriol 179, 7321–7330.
Shevchik, V. E., Boccara, M., Vedel, R. & Hugouvieux-Cotte-Pattat, N. (1998). Processing of the pectate lyase PelI by extracellular proteases of Erwinia chrysanthemi 3937. Mol Microbiol 29, 1459–1469.[CrossRef][Medline]
Shevchik, V. E., Condemine, G., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1999a). The exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937. J Bacteriol 181, 3912–3919.
Shevchik, V. E., Kester, H. C. M., Benen, J. A. E., Visser, J., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1999b). Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937. J Bacteriol 181, 1652–1663.
Soriano, M., Blanco, A., Diaz, P. & Pastor, F. I. J. (2000). An unusual pectate lyase from a Bacillus sp. with high activity on pectin: cloning and characterization. Microbiology 146, 89–95.
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T-7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113–130.[CrossRef][Medline]
Tamaru, Y. & Doi, R. H. (2001). Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome. Proc Natl Acad Sci U S A 98, 4125–4129.
Tardy, F., Nasser, W., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1997). Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors. J Bacteriol 179, 2503–2511.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Vellanoweth, R. L. (1993). Translation and its regulation. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, pp. 699–711. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
Yoder, M. D., Keen, N. T. & Jurnak, F. (1993). New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science 260, 1503–1507.
Received 4 October 2005;
revised 15 November 2005;
accepted 23 November 2005.
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