Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK1
Author for correspondence: Harry J. Flint. Tel: +44 1224 716651. Fax: +44 1224 716687. e-mail: h.flint{at}rri.sari.ac.uk
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ABSTRACT |
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Keywords: esterase, cellulosome, Ruminococcus, rumen, dockerin
The GenBank accession numbers for the sequences reported in this paper are AJ238716 (cesA) and AJ272430 (xynE).
a Present address: CNR-IABBAM, Via Argine 1085-80147 Ponticelli-Napoli, Italy.
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INTRODUCTION |
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The rumen is a particularly active site for anaerobic breakdown of a variety of plant cell wall material. Among ruminal micro-organisms, phenolic acid esterase activities have been reported from anaerobic fungi (Borneman et al., 1991 , 1992
), and a gene encoding a phenolic acid esterase has been isolated from Butyrivibrio fibrisolvens (Dalrymple et al., 1996
). Acetylxylan esterases have been reported from rumen fungi (Dalrymple et al., 1997
) and from the rumen bacteria Butyrivibrio fibrisolvens and Fibrobacter succinogenes (McDermid et al., 1990
; Hespell & OBryan-Shah, 1988
). There is little evidence so far for the involvement of esterase activities in cell wall degradation by Ruminococcus spp. (Akin et al., 1993
), which represent one of the most numerous groups of cellulolytic bacteria in the rumen. Although it has been shown that Ruminococcus albus can release phenolic monomers from plant material (Giraud et al., 1997
), there have been no biochemical or molecular studies on esterases from ruminococci. We report here the specificities and relationships of two catalytic domains from Ruminococcus flavefaciens enzymes that function as acetylesterases, and also identify a third gene that encodes a multidomain esterase. Recent evidence indicates that most plant cell wall polysaccharidases in R. flavefaciens 17 carry dockerin sequences, indicating that they are likely to belong to cellulosome complexes (Kirby et al., 1997
). The three R. flavefaciens multidomain esterases discussed here, two of which carry xylanase domains within the same polypeptide, also carry dockerin-like sequences, suggesting that they may also be cellulosome-associated.
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METHODS |
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xynE was identified initially by PCR from chromosomal DNA using the oligonucleotide JIIf [CCI(C/T)TI(A/G)TIGA(A/G)TA(T/C)TA(T/C)ATIGTIG] (Kirby, 1996 ) as a family 11 domain-specific primer [corresponding to the amino acid sequence P(L/F)(V/I/M)EYY(M/I)V] together with the reverse primer pESTr (CCICCCATIGARAAICC), designed to recognize coding regions (corresponding to the amino acids GFSMGG) of related family 1 esterases.
Construction of pXynB.est, expressing the esterase domain of XynB.
A 965 bp fragment was recovered from the plasmid clone X1022, which carried the C-terminal coding sequences of the xynB gene (Zhang et al., 1994 ; Zhang, 1992
). Insertion of this fragment into the SmaI site of pUC18, in the appropriate orientation, resulted in an in-frame translational fusion with the start of the lacZ gene. The predicted product carried the first 10 amino acid residues of the lacZ product followed by residues 435755 of the XynB polypeptide. The XynB residues correspond to the last 28 amino acids of a conserved stabilizing-type domain, the putative family 3 esterase domain and the first 54 (out of 82) amino acids from the C-terminal XynB dockerin region (Kirby et al., 1997
). For clarity, this fusion construct is refered to here as pXynB.est, but was previously designated XBest (Kirby et al., 1998
). DNA manipulations and hybridization procedures followed standard protocols (Sambrook et al., 1989
).
DNA sequence analyses.
DNA sequences were determined on both strands using an ABI377 automated sequencer and appropriate oligonucleotide primers. Computer analysis was done with the UWGCG software available through the Daresbury Seqnet and HGMP facilities (UK). Multiple alignments were done using CLUSTAL V or W, and phylogenetic analysis through the PHYLIP package.
Acetylxylan esterase assay.
Escherichia coli DH5 or XL-1 Blue cells carrying the plasmid clones pCesA.est and pXynB.est were pelleted by centrifuging for 10 min at 2000 g, 4 °C (Sorvall SS34). The pellet was washed with 10 ml buffer A (50 mM sodium phosphate buffer pH 6·5, 2 mM DTT). The pellet was finally resuspended in 2 ml of this buffer. The cells were broken by sonication in a Soniprep 150 (SANYO Gallenkamp PLC), using a 12 µm Exponential Microprobe with three strokes of 1 min each, cooling on ice. The sonicate was used for the enzyme determinations. Assays were performed in sodium phosphate buffer (50 mM) at pH 6·8 in the presence of 1% acetylated birchwood xylan: either native, steam-extracted xylan (supplied by J. Puls, BFH, Hamburg, Germany) or xylan chemically acetylated by the method of Johnson et al. (1988)
. The acetate released was analysed by HPLC using an Aminex HPX-87H Bio-Rad column (300x7·8 mm) and a refractive index detector. Samples were eluted with 4 mM H2SO4 at 0·6 ml min-1 and 35 °C. D-Fucose was used as an internal standard. Reducing sugar release was determined by the method of Lever (1977)
.
SDS-PAGE and acetylesterase zymogram.
SDS-PAGE was based on the method described by Laemmli (1970) . Protein samples were denatured by boiling for 5 min in SDS loading buffer (final concentration: 50 mM Tris/HCl pH 6·8, 100 mM DTT, 2% SDS, 10% glycerol, 0·1% bromophenol blue) before applying to the gel. Molecular masses were estimated by comparison with molecular mass standards (Sigma). After electrophoresis the gel was washed in 50 mM Tris/HCl pH 7·5 and stained for acetylesterase activity in 50 mM Tris/HCl pH 7·5 containing 0·05 mg ß-naphthyl acetate ml-1 and 0·05 mg Diazo Blue ml-1. Coomassie blue was used to stain protein bands. Growth of R. flavefaciens was as described previously (Flint et al., 1993
).
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RESULTS |
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Relationships of dockerin-like regions from R. flavefaciens esterases
Five dockerin sequences have now been identified in enzymes of R. flavefaciens 17, including those from the CesA, XynE and XynB multidomain esterases (Fig. 4). All five include at least one potential calcium-binding motif, and show limited homology with dockerins from cellulolytic Clostridium spp. (Bayer et al., 1998a
). A phylogenetic comparison including typical type I (LicB) and type II (CipA) dockerins from C. thermocellum is shown in Fig. 5
. It is clear that the dockerins from the two newly reported esterases CesA and XynE diverge significantly from those of XynB, XynD and EndA. More specifically, they differ at residues 10 and 11 within their putative calcium-binding motifs (Fig. 4
); these two residues have been proposed by Pages et al. (1997)
to be particularly important in cohesin-binding selectivity of dockerins from Clostridium spp. The R. flavefaciens dockerins do not cluster closely with C. thermocellum dockerins of type I or type II, and appear to represent a distinct group, or groups (Fig. 5
).
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DISCUSSION |
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The exact roles of the XynB, XynE and CesA esterases in plant cell wall breakdown have yet to be fully established. We have shown that the XynB and CesA activities are capable of removing acetyl groups from acetylated xylans. Since neither domain requires the simultaneous presence of an endoxylanase, both enzymes appear capable of acting on the polysaccharide, although the slight enhancement of acetate release in the presence of xylanase suggests that they may be more active on oligosaccharides than on the polysaccharide. Conversely, the slight stimulation of reducing sugar release by a cloned R. flavefaciens xylanase in the presence of the acetylesterase suggests that the removal of acetyl groups makes the substrate more accessible to xylanase hydrolysis. The association of the XynB esterase with a family 11 xylanase in the same polypeptide makes it highly plausible that deacetylation of xylans is the primary role of this domain. The role of the CesA enzyme, however, may be quite different and the function of its C-terminal domain has yet to be established. The lack of detectable deacetylating activity against sugar beet pulp does not rule out a role of the N-terminal domain of CesA in deacetylation of particular regions of pectins or pectin breakdown products (Searle-van Leeuwen et al., 1992 ; Shevchik & Hugouvieux-Cotte-Pattat, 1997
) and this possibility deserves further study. The specificity of the esterase domain identified in XynE has yet to be investigated.
In general, esterase domains present in carbohydrate-active enzymes are quite diverse with respect to their primary amino acid sequences and are currently grouped into at least seven different families (see http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html). The acetylesterase domains from R. flavefaciens 17 XynB and CesA appear to be the first active esterase domains belonging to family 3 to be reported in bacterial enzymes. The putative esterase domain identified here in R. flavefaciens 17 XynE, on the other hand, belongs to family 1. Family 1 domains have been reported previously in multidomain xylanases from several bacterial species and include enzymes that show both acetyl-esterase and phenolic acid esterase activities (Bartolomé et al., 1997 ). In addition, NodB-homologous domains belonging to esterase family 4, which can show acetylesterase activity (Laurie et al., 1997
), have been found in enzymes from Cellulomonas fimi, Cellvibrio mixtus and Streptomyces lividans (Shareck et al., 1995
), and in the XynA xylanase from Ruminococcus albus (database accession number U43089: T. Nagamine and co-workers). To date, therefore, representatives of three different esterase families (1, 3 and 4) have been shown to occur in xylanases from Ruminococcus spp.
In addition to their catalytic domains, the three R. flavefaciens esterases considered here all carry dockerin-like regions. These dockerins are assumed to be involved in binding to corresponding cohesin domains present in a scaffolding protein or proteins in a cellulosome multienzyme complex (Bayer et al., 1998b ) since evidence has recently been obtained for a scaffolding protein in R. flavefaciens 17 (Ding et al., 1999
; S.-Y. Ding and others, unpublished). The likely occurrence of esterase activities in addition to hydrolytic activities in cellulosomal complexes has been suggested elsewhere for Clostridium thermocellum (Blum et al., 1998
). Interestingly, we show here that the CesA and XynE dockerins diverge significantly in their sequences from each other and from those reported previously in XynB, EndA and XynD, including differences in the amino acid positions implicated in the cohesin selectivity of clostridial dockerins (Pages et al., 1997
). This divergence raises the possibility that more than one dockerincohesin specificity might be involved in the assembly of the putative R. flavefaciens cellulosome complexes. CesA and XynE might, for example, bind to different cohesins compared with XynB, XynD and EndA, with consequences for the temporal and spatial positioning of these polypeptides on the cell surface. In C. thermocellum only one type of dockerincohesin interaction is involved in the binding of enzyme subunits to the scaffolding protein, but a second is involved in attachment of the complex to cell surface anchoring proteins (Salamitou et al., 1994
; Bayer et al., 1998a
). Understanding of the functional significance of the dockerin divergence seen in R. flavefaciens enzymes must await the outcome of detailed studies on dockerincohesin interactions in this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bartolomé, B., Faulds, C. B., Kroon, P. A., Waldron, K., Gilbert, H. J., Hazlewood, G. P. & Williamson, G. (1997). An Aspergillus niger esterase (FAE-III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (XYLD) release 5,5'-ferulic dehydroodimer (diferulic acid) from barley and wheat cell walls.Appl Environ Microbiol 63, 208-212.[Abstract]
Bayer, E. A., Morag, E., Lamed, R., Yaron, S. & Shoham, Y. (1998a). Cellulosome structure: four-pronged attack using biochemistry, molecular biology, crystallography and bioinformatics. In Carbohydrases from Trichoderma reesei and Other Microorganisms, pp. 39-65. Edited by M. Claeyssens, W. Nerinckx & K. Piens. Cambridge: Royal Society of Chemistry.
Bayer, E. A., Shimon, L. J. W., Shoham, Y. & Lamed, R. (1998b). Cellulosomes structure and ultrastructure.J Struct Biol 124, 221-234.[Medline]
Biely, P., MacKenzie, C. R., Puls, J. & Schneider, H. (1986). Cooperativity of esterases and xylanases in enzymatic degradation of acetylxylan.Bio/Technology 4, 731-733.
Blum, D. L., Kataeva, I., Li., X.-L. & Ljungdahl, L. G. (1998). Phenolic acid esterase activity of Clostridium thermocellum cellulosome is attributed to previously unknown domains of XynY and XynZ. In Genetics, Biochemistry and Ecology of Cellulose Degradation (MIE Bioforum 98), pp. 478. Edited by K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita & T. Kimura. Tokyo: UNI Publishers.
Borneman, W. S., Ljungdahl, L. G., Hartley, R. D. & Akin, D. E. (1991). Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.Appl Environ Microbiol 57, 2337-2344.[Medline]
Borneman, W. S., Ljungdahl, L. G., Hartley, R. D. & Akin, D. E. (1992). Purification and partial characterisation of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC2.Appl Environ Microbiol 58, 3762-3766.[Abstract]
Christov, L. P. & Prior, B. A. (1993). Esterases of xylan degrading microorganisms: production, properties and significance.Enzyme Microb Technol 15, 460-475.[Medline]
Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K. & Doctor, B. P. (1993). Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.Protein Sci 2, 366-382.
Dalrymple, B. P., Swadling, Y., Cybinski, D. H. & Xue, G.-P. (1996). Cloning of a gene encoding cinnamoyl ester hydrolase from the ruminal bacterium Butyrivibrio fibrisolvens E14 by a novel method.FEMS Microbiol Lett 143, 115-120.[Medline]
Dalrymple, B. P., Cybinski, D. H., Layton, I., McSweeney, C. S., Xue, G.-P., Swadling, Y. J. & Lowry, J. B. (1997). Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases.Microbiology 143, 2605-2614.[Abstract]
Ding, S.-Y., Bayer, E. A., Shoham, Y., Lamed, E., McCrae, S. I., Kirby, J., Aurilia, V. & Flint, H. J. (1999). Preliminary evidence of high molecular weight scaffoldin-like proteins from Ruminococcus flavefaciens. 3rd Carbohydrate Bioengineering Meeting, Newcastle, UK, abstract 5.12.
Flint, H. J., Martin, J. & McPherson, G. A. (1993). A bifunctional enzyme, with separate xylanase and ß(1,31,4)glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens.J Bacteriol 175, 2943-2951.[Abstract]
Gerngross, V. T. & Demain, A. L. (1993). Sequencing of a Clostridium thermocellum gene cipA encoding the cellulosomal SL protein reveals an unusual degree of internal homology.Mol Microbiol 8, 325-334.[Medline]
Giraud, I., Besle, J. M. & Fonty, G. (1997). Hydrolysis and degradation of esterified phenolic acids from the maize cell wall by rumen microbial species. Reprod Nutr Dev Suppl 5253.
Grépinet, O., Chebrou, M.-C. & Béguin, P. (1988). Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum.J Bacteriol 170, 4582-4588.[Medline]
Hespell, R. B. & OBryan-Shah, P. J. (1988). Esterase activities in Butyrivibrio fibrisolvens.Appl Environ Microbiol 54, 1917-1927.[Medline]
Iiyama, K., Lam, T. P. T. & Stone, B. A. (1994). Covalent cross-links in the cell wall.Plant Physiol 104, 315-320.
Johnson, K. G., Fontana, J. D. & MacKenzie, C. R. (1988). Measurement of acetyl xylan esterase in Streptomyces.Methods Enzymol 160, 552-560.
Kirby, J. (1996). Multiplicity and organisation of plant cell wall degrading enzymes in Ruminococcus flavefaciens. PhD thesis, University of Aberdeen.
Kirby, J., Martin, J. C., Daniel, A. S. & Flint, H. J. (1997). Dockerin-like sequences from the rumen cellulolytic bacterium Ruminococcus flavefaciens.FEMS Microbiol Lett 149, 213-219.[Medline]
Kirby, J., Aurilia, V., McCrae, S. I., Martin, J. C. & Flint, H. J. (1998). Plant cell wall degrading enzyme complexes from the cellulolytic rumen bacterium Ruminococcus flavefaciens.Biochem Soc Trans 26, S169.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680-685.[Medline]
Laurie, J. I., Clarke, J. H., Ciruela, A., Faulds, C. B., Williamson, G., Gilbert, H. J., Rixon, J. E., Millward-Sadler, J. & Hazlewood, G. P. (1997). The nodB domain of a multidomain xylanase from Cellulomonas fimi deacetylates acetylxylan.FEMS Microbiol Lett 148, 261-264.
Lever, M. (1977). Carbohydrate determination with 4-hydroxybenzoic acid hydrazide (PAHBAH): effect of bismuth on the reaction.Anal Biochem 81, 21-27.[Medline]
McDermid, K. P., MacKenzie, C. R. & Forsberg, C. W. (1990). Esterase activities of Fibrobacter succinogenes S85.Appl Environ Microbiol 56, 127-132.
Pages, S., Belaich, A., Belaich, J.-P., Morag, E., Lamed, R., Shoham, Y. & Bayer, E. A. (1997). Species specificity of the cohesindockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain.Proteins Struct Funct Genet 29, 517-527.[Medline]
Rombouts, F. M. & Thibault, J. F. (1986). Sugar beet pectins: chemical structure and gelation through oxidative coupling. Chemistry and function of pectins. ACS (Am Chem Soc) Symp Ser 310, 4960.
Salamitou, S., Raynaud, O., Lemaire, M., Coughton, M., Béguin, P. & Aubert, J. P. (1994). Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome integrating protein CipA.J Bacteriol 176, 2822-2827.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schimming, S., Schwarz, W. H. & Staudenbauer, W. L. (1992). Structure of the Clostridium thermocellum gene licB and the encoded ß-1,31,4-glucanase.Eur J Biochem 204, 13-19.[Abstract]
Searle-van Leeuwen, H. J. F., van den Brock, H., Schols, H. A., Beldman, G. & Voragen, A. G. J. (1992). Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regimes of pectins.Appl Microbiol Biotechnol 38, 347-349.
Shareck, F., Biely, P., Morosoli, R & Kluepfel, D. (1995). Analysis of DNA flanking the xynB locus of Streptomyces lividans reveals genes encoding acetyl xylan esterase and the RNA component of RnaseP.Gene 153, 105-109.[Medline]
Shevchik, V. E. & Hugouvieux-Cotte-Pattat, N. (1997). Identification of a bacterial pectin acetylesterase in Erwinia chrysanthemi 3937.Mol Microbiol 24, 1285-1301.[Medline]
Upton, C. & Buckley, J. T. (1995). A new family of lipolytic enzymes?Trends Biochem Sci 20, 178-179.[Medline]
Williamson, G., Kroon, P. A. & Faulds, C. R. (1998). Hairy plant polysaccharides: a close shave with microbial esterases.Microbiology 144, 2011-2023.
Wood, T. M. & McCrae, S. I. (1986). The effect of acetyl groups on the hydrolysis of ryegrass cell walls by xylanase and cellulase from Trichoderma koningii.Phytochemistry 25, 1053-1055.
Zhang, J.-X. (1992). Genetic determination of xylanase in the rumen bacterium Ruminococcus flavefaciens. PhD thesis, University of Aberdeen.
Zhang, J.-X., Martin, J. & Flint, H. J. (1994). Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens.Mol Gen Genet 245, 260-264.[Medline]
Received 27 September 1999;
revised 5 March 2000;
accepted 13 March 2000.