Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences

Vincenzo Auriliaa,1, Jennifer C. Martin1, Sheila I. McCrae1, Karen P. Scott1, Marco T. Rincon1 and Harry J. Flint1

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


   ABSTRACT
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INTRODUCTION
METHODS
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DISCUSSION
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Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

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.


   INTRODUCTION
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INTRODUCTION
METHODS
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DISCUSSION
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Efficient breakdown of plant cell wall material by micro-organisms requires not only glycosidase activities that cleave polysaccharide chains, but also activities that remove groups that are connected by ester linkages to polysaccharides (Christov & Prior, 1993 ; Williamson et al., 1998 ). Xylans are often heavily substituted with acetyl groups and can also be connected to phenolic acids, and hence to lignin, via ester linkages involving arabinose substituents (Iiyama et al., 1994 ), while pectins carry esterified methyl and acetyl groups (Rombouts & Thibault, 1986 ; Searle-van Leeuwen et al., 1992 ). There is evidence that acetyl substituents can limit the attack of endoxylanases upon xylans (Wood & McCrae, 1986 ; Biely et al., 1986 ; Puls et al., 1991) and ferulic acid ester linkages may represent a significant impediment to enzymic removal of polysaccharides from lignified plant cell walls (Iiyama et al., 1994 ).

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 & O’Bryan-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.


   METHODS
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Gene isolation.
About 2000 clones from a R. flavefaciens {lambda}EMBL3 phage library were screened by detection of acetylesterase activity. The phages were transferred to Whatman no.1 filter paper and incubated with ß-naphthyl acetate (0·5 mg ml-1) and tetrazotized o-dianisidine (Blue Diazo) in 50 mM sodium phosphate buffer pH 7·4. Two positive clones were isolated and shown to be identical in their restriction enzyme profiles. The clone pCesA.est is a plasmid subclone from one phage that was subsequently shown to carry the coding region for the N-terminal 390 amino acids of the cesA gene product. Probes derived from pCesA.est were used to obtain overlapping plasmid clones by screening a pUCI3 plasmid library of R. flavefaciens 17 in order to obtain the complete cesA gene sequence. Correspondence between the cloned sequences and the chromosomal cesA gene was confirmed by direct PCR amplification using a primer set internal to cesA.


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 435–755 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{alpha} 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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Zymogram analysis of acetylesterases from R. flavefaciens 17
The production of acetylesterases by cultures of R. flavefaciens 17 was confirmed by zymogram analysis. Supernatant proteins from cultures grown on oat-spelt xylan were separated by SDS-PAGE and tested for activity against ß-naphthyl acetate after renaturation as shown in Fig. 1. The predominant band of acetylesterase activity was around 80 kDa, with minor bands at 90 and 35 kDa.



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Fig. 1. Acetylesterases from culture supernatant of R. flavefaciens 17. Polypeptides from the supernatant of a culture grown with oat-spelt xylan as substrate were separated by SDS-PAGE, followed by renaturation and detection of esterase activity against ß-naphthyl acetate (see Methods).

 
Isolation of the R. flavefaciens cesA gene encoding a new multidomain acetylesterase
An acetylesterase domain was identified previously in the 90 kDa multidomain xylanase XynB (Kirby et al., 1997 ). In order to isolate further genes encoding acetylesterases, a lambda phage library of R. flavefaciens 17 chromosomal DNA was screened for plaques showing activity against ß-naphthyl acetate. Active plasmid clones were obtained after subcloning from one positive phage clone, and the sequence of a new gene responsible for acetylesterase activity, designated cesA, was completed after isolation of further overlapping clones obtained by hybridization screening of a pUC13 plasmid library (see Methods). The predicted product of the cesA gene is a 768 amino acid polypeptide starting with a putative signal peptide sequence (40 amino acids) and a predicted mature molecular mass of approximately 84 kDa (cesA accession number AJ238716; Fig. 2). The domain that follows the signal peptide shares 44% amino acid identity with the acetylesterase domain of R. flavefaciens 17 XynB, and 34% amino acid identity with the acetylesterase domain of BnaC from the rumen anaerobic fungus Neocallimastix patriciarum (Zhang et al., 1994 ; Dalrymple et al., 1997 ; Kirby et al., 1997 ) (Fig. 3). This indicates that the CesA and XynB domains both belong to carbohydrate esterase family 3, whose members show features in common with lipases (Dalrymple et al., 1997 ; Upton & Buckley, 1995 ; see also http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html). The esterase domain in CesA is followed by a region of approximately 80 amino acids that resembles dockerins found previously in three R. flavefaciens polysaccharidases (Kirby et al., 1997 ). Finally CesA carries a C-terminal domain (418 amino acids) of unknown function that showed no significant matches with other proteins in database searches.



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Fig. 2. (a) Domain structures of CesA, XynB and XynE from Ruminococcus flavefaciens 17. (b) Truncated products encoded by plasmids pCesA.est (C-terminal truncation of CesA) and pXynB.est (N-terminal truncation of XynB) (see Methods).

 


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Fig. 3. Multiple alignment (CLUSTAL V) of acetylesterase domains of CesA, XynB and Neocallimastix patriciarum BnaC. The three amino acids thought to be involved in catalysis in enzymes of this family (Cygler et al., 1993 ) (see text) are shown in bold and marked {bullet} above the sequence.

 
Evidence for an additional multidomain xylanase, XynE, carrying a family 1 esterase catalytic domain
An additional new gene, designated xynE, was identified in R. flavefaciens 17, initially by amplification of a 1·5 kb chromosomal DNA sequence using a domain-specific primer designed for family 11 xylanases (Kirby, 1996 ) in conjunction with a primer designed to recognize family 1 esterases. The complete XynE product (792 amino acids, accession number AJ272430) shows an N-terminal signal peptide sequence followed by a region between residues 26 and 421 of very high homology (99·5% amino acid identity) with XynB, corresponding to the family 11 xylanase and adjacent ‘stabilizing’ domains (Fig. 2). Thereafter XynE carries a dockerin-like sequence and a threonine-rich linker, followed by a C-terminal domain of 277 amino acid residues that shares 42% amino acid identity with a family 1 esterase domain from Clostridium thermocellum XynZ (Grépinet et al., 1988 ) and 79% amino acid identity with an unknown portion of xylanase 1 from a Ruminococcus sp. (accession number Z49970: C. Arakaki and co-workers).

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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Fig. 4. Dockerin-like regions from R. flavefaciens CesA and XynE compared with those from R. flavefaciens EndA, XynB and XynD. Identical residues conserved in the R. flavefaciens enzymes and in dockerins from Clostridium thermocellum LicB (Schimming et al., 1992 ) and CipA (Gerngross & Demain, 1993 ) are indicated in bold. Asterisks indicate residues corresponding to the conserved positions 1, 3, 5, 9 and 12 identified in the calcium-binding motifs (shown by the solid line and flanking spaces) of C. thermocellum and C. cellulolyticum dockerins by Pages et al. (1997) .

 


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Fig. 5. Phylogenetic tree showing the relationships of five dockerins from R. flavefaciens 17 enzymes (CesA, XynE, XynB, XynD and EndA) and typical type I (LicB) and type II (CipA) dockerins from Clostridium thermocellum. A variable 8 amino acid region between the two conserved dockerin motifs in XynB and XynD (Fig. 4) was omitted from the analysis. The tree shown is an unrooted neighbour-joining tree.

 
Specificity of acetylesterase domains from enzymes of R. flavefaciens 17
The specificities of the acetylesterase domains from XynB and CesA, expressed in E. coli, were studied in truncated subclones that lack the N-terminal xylanase domain, in the case of XynB, or the unknown C-terminal domain in the case of CesA (Fig. 2, Table 1). Both domains showed greater activity against ß-naphthyl acetate than against ß-naphthyl propionate, although the relative activities against ß- and {alpha}-naphthyl acetate differed. Neither enzyme showed activity against methyl ferulate or against MUTMAC [4-methylumbelliferyl-7-(p-trimethylammonium cinnamate) chloride], indicating that they lack ferulic acid esterase activity. Both enzymes were able to release acetate from chemically acetylated xylan (55·3 and 27·8 µmol acetate released per mg protein for XynB and CesA, respectively, in 24 h incubations) or from native, steam-extracted xylan (92·8 and 54·7 µmol acetate released per mg protein for XynB and CesA, respectively, in 24 h). Neither enzyme was able to release detectable amounts of acetate from sugar beet pulp (results not shown).


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Table 1. Activity of extracts from E. coli cells carrying pXynB.est or pCesA.est upon soluble esterase substrates

 
The possibility of synergy between the debranching activity of the acetylesterase and cleavage of the main xylan chain by xylanases was studied with native steam-extracted acetylated xylan as substrate (Table 2). Xylanase activity was provided by the cloned R. flavefaciens xylanase XynD, whose N-terminal xylanase domain shares 95% sequence identity with the corresponding domain from XynB (Flint et al., 1993 ). A small degree of synergy (approx. 50%) was observed with respect to acetate release for each of the esterase domains acting with the xylanase, indicating that the esterases may be slightly more active in deacetylating xylooligosaccharides than the polysaccharide. A small degree of synergy was also observed with respect to reducing sugar release, consistent with some interference of xylanase action by acetyl substituents (Biely et al., 1986 ; Wood & McCrae, 1986 ). The degree of acetylation of the steam-extracted xylan used here was approximately 15%, and it is possible that greater degrees of synergy would be observed with more heavily substituted substrates.


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Table 2. Action of isolated acetylesterase and xylanase domains upon steam-extracted birchwood xylan

 
The specificity of the XynE esterase domain has not yet been determined.


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This work establishes for the first time that Ruminococcus spp. possess esterases that are likely to play an important role in plant cell wall degradation in the rumen. More specifically, we have identified three genes in R. flavefaciens 17 whose products contain esterase domains: XynB (predicted mature size 87 kDa), CesA (84 kDa) and XynE (88 kDa).

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 dockerin–cohesin 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 dockerin–cohesin 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 dockerin–cohesin interactions in this species.


   ACKNOWLEDGEMENTS
 
This work was supported by the Scottish Executive Rural Affairs Department. We are grateful for support via the EU TMR Fellowship scheme to V.A. We are also grateful to Jurgen Puls for the kind gift of steam-extracted birch wood xylan and to Brian Dalrymple, Gary Williamson and Ed Bayer for valuable discussion.


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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 27 September 1999; revised 5 March 2000; accepted 13 March 2000.