(Received for publication, August 1, 1995; and in revised form, September 20, 1995)
From the
Two cDNAs, designated xynA and manA, encoding
xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated
from a cDNA library derived from mRNA extracted from the anaerobic
fungus, Piromyces. XYLA and MANA displayed properties typical
of endo-1,4-xylanases and mannanases, respectively. Neither enzyme
hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818
base pairs, respectively, coding for proteins of M
68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of
MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family
26. A 40-residue reiterated sequence, which was homologous to
duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was
located at the C terminus of MANA. XYLA contained two regions that
exhibited sequence identity with the catalytic domains of glycosyl
hydrolase Family 11 xylanases and were separated by a duplicated
40-residue sequence that exhibited strong homology to the C terminus of
MANA. Analysis of truncated derivatives of MANA confirmed that the
N-terminal 458-residue sequence constituted the catalytic domain, while
the C-terminal domain was not essential for the retention of catalytic
activity. Similar deletion analysis of XYLA showed that the C-terminal
catalytic domain homologue exhibited catalytic activity, but the
corresponding putative N-terminal catalytic domain did not function as
a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence
conserved in XYLA and MANA to glutathione S-transferase,
generated a hybrid protein that did not associate with cellulose, but
bound to 97- and 116-kDa polypeptides that are components of the
multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this
domain in the assembly of the enzyme complex is discussed.
Endo-1,4-xylanase (xylanase; EC 3.2.1.8) and
endo-
1,4-mannanase (mannanase; EC 3.2.1.78) hydrolyze,
respectively, the
1,4-linked polysaccharide backbones of xylans
and mannans, which form the two major hemicellulose components of
hardwoods and softwoods (1) . Recent studies on the structure
of xylanases have revealed that some enzymes are comprised of single
catalytic domains while other xylanases are modular, consisting of
single or multiple catalytic domains fused via linker sequences to
noncatalytic sequences, some of which constitute cellulose binding
domains (CBD(
); (2) and (3) ).
Hemicellulases derived from aerobic microorganisms do not appear to
associate, while anaeorbic organisms often synthesize multienzyme
cellulase-hemicellulase complexes(2) . Many xylanases have been
analyzed, but their catalytic domains appear to have evolved from only
two progenitor sequences. In contrast, the primary structures of only
seven mannanases have been determined, to date. Based on sequence
alignments, two of the enzymes belong to glycosyl hydrolase Family 5 (4, 5) , three of the mannanases are located in Family
26(6, 7, 8) , while the two enzymes from
aerobic fungi exhibit significant sequence identity, although they have
not been assigned to a specific family (9) . The complexity of
the molecular architecture of these enzymes is variable; the Streptomyces lividans, Bacillus spp., and Pseudomonas
fluorescens subsp. cellulosa(4, 6, 8) mannanases consist of single
catalytic domains, while the corresponding Caldocellum
saccharolyticum enzyme (5) is comprised of two catalytic
domains, that exhibit mannanase and endo-
1,4-glucanase activity,
respectively, and are separated by a duplicated CBD homologue.
Recent studies in our laboratories have focussed on plant cell wall-degrading enzymes of anaerobic fungi that are particularly active against the more recalcitrant plant structural polysaccharides. These organisms produce cellulases and hemicellulases that associate into large molecular weight multienzyme complexes and bind tightly to cellulose(10, 11) . Recently, the molecular architecture of cellulases and xylanases from Neocallimastix patriciarum has been analyzed. Two of the enzymes contain catalytic domains that are linked to a duplicated 40 residue noncatalytic C-terminal domain (12, 13) of unknown function. In general, there is a paucity of information on the structure/function relationship of plant cell wall hydrolases of rumen chytridiomycetes. Specifically, it remains to be established whether the 40-residue noncatalytic domain is conserved between different species of fungi and if so, whether the formation of cellulase/hemicellulase complexes in these organisms is mediated by a common mechanism involving this sequence; in addition, the molecular architecture of rumen fungal plant cell wall hydrolases, other than cellulases and xylanases, remains to be elucidated, and it is unclear to what extent the plant cell wall degrading systems in these organisms arose through horizontal gene transfer between rumen fungi. To address these questions we have isolated cDNAs encoding plant cell wall hydrolases from another rumen fungus, Piromyces, and we have used the cloned sequences to characterize the encoded enzymes. In this report we describe the molecular architecture and biochemical properties of a xylanase and mannanase from Piromyces. Both enzymes contain the same noncatalytic reiterated 40-residue sequence previously observed in Neocallimastix enzymes. The mannanase exhibits homology with the mannanases from Bacillus spp. and from P. fluorescens subsp. cellulosa. The xylanase contains a large duplicated domain which shows sequence identity with glycosyl hydrolase Family 11 xylanases, although only one of these domains is functional. The conserved noncatalytic 40-residue reiterated sequence, which does not bind to cellulose, binds specifically to polypeptides of 97 and 116 kDa that are components of the multienzyme cellulose-binding complexes produced by Piromyces and N. patriciarum, respectively. The possible role of this sequence as a protein docking domain that mediates assembly of the anaerobic fungal multienzyme complex is discussed.
To sequence xynA and manA, a series of nested deletions of each of the two cDNAs was constructed using the exonuclease III/S1-nuclease kit supplied by Promega. The nested deletions were sequenced using a PRISM(TM) ready reaction dye-deoxy terminator cycle sequencing kit supplied by Applied Biosystems. Sequences were compiled and ordered using the computer programs of Staden(18) . The two cDNAs were sequenced completely in both strands.
Figure 1:
Restriction map of xynA and manA. The restriction maps of xynA and manA are shown in A and B, respectively. The
positions of the cleavage sites for the restriction enzymes HindIII (H), EcoRI (R), EcoRV (Rv), KpnI (K), NcoI (N), PvuII (P), StuI (St), ScaI (Sc), SstI (Ss), and XhoI (X) are shown. The location of the sequences
encoding the signal peptides (), catalytic domains (
), 40
residue non-catalytic reiterated sequences (&cjs2108;) and linker
sequences (&cjs2112;) of XYLA and MANA are shown. The plasmids in which
the 3` region of xynA and manA have been deleted by
exonuclease III/S1-nuclease digestion are designated pGX8-10 and
pGM2-4, respectively. The positions of the 3` nucleotides of
truncated xynA and manA in these plasmids are shown
in the nucleotide sequences of the two cDNAs depicted in Fig. 3.
The cDNA inserts containing 5` deletions were cloned into appropriate
plasmids such that the xynA and manA derivatives were
in-frame with the vectors' lacZ` translational starts.
The capacity of the plasmids to express a functional hemicellulase is
expressed as a percent of the activity of the respective full-length
enzymes.
Figure 3: Sizes of recombinant XYLA and MANA. Cell-free extracts derived from E. coli JM83 harboring pGX1 (A) and pGM1 (B) were subjected to SDS-PAGE using 10% (w/v) polyacrylamide gels containing soluble xylan or carob galactomannan, respectively. Zymogram analysis was conducted as described under ``Materials and Methods.'' Sizes of standard proteins and the largest polypeptides that exhibited xylanase (A) and mannanase (B) activity, respectively, are shown in kilodaltons.
MANA hydrolyzed carob and locust bean galactomannan and ivory nut mannan. The major products released from each substrate were mannose and mannobiose. The enzyme exhibited no activity against other plant structural polysaccharides (Table 1). To evaluate whether MANA had a preference for polymeric substrates, the rate at which the enzyme hydrolyzed a range of mannooligosaccharides was evaluated. The data (Table 2) showed that the enzyme had very high activity against mannohexaose compared with mannopentaose and smaller mannooligosaccharides. The major products generated when the reaction had gone to completion were mannose and mannobiose (data not shown). MANA displayed no detectable transglycosylating activity. These data indicate that the active site of MANA has a minimum of six sugar-binding sites and that substrates which occupy five or less of these sites exhibit low affinity for the enzyme. This is in contrast to the mannanase from P. fluorescens subsp. cellulosa which displays similar activity toward mannohexaose, mannotetraose, and mannotriose(6) . In general, the biochemical properties of XYLA and MANA are typical of xylanases and mannanases, respectively.
Figure 2: Nucleotide sequences of xynA and manA. The nucleotide sequences of xynA and manA and the deduced primary structures of the encoded enzymes are shown in A and B, respectively. The proposed linker sequences of XYLA and MANA are boxed.
Between the two putative catalytic domains of XYLA is a 40-residue sequence which is tandemly repeated (Fig. 2). The reiterated sequence exhibits extensive sequence identity with duplicated noncatalytic domains located at the C terminus of CelB and XYLA of N. patriciarum(12, 13) and with a triplicated sequence from Piromyces MANA (Fig. 4). The overall identity between the 40-residue sequences is 38%.
Figure 4: Comparison of the reiterated 40-residue sequences found in cellulases and hemicellulases from N. patriciarum and Piromyces. The position of the first residue of each reiterated amino acid sequence within the primary structure of N. patriciarum XYLA(12) , N. patriciarum CelB(13) , Piromyces XYLA and Piromyces MANA is shown. Those residues which exhibit identity or similarity in at least five of the sequences are boxed.
The N-terminal
portion of MANA comprised of a 450-residue domain that exhibits
significant homology between residues 180 and 382 to the catalytic
domains of two mannanases from Bacillus spp., mannanase A from P. fluorescens subsp. cellulosa and an endoglucanase
from Bacteroides ruminicola (Fig. 5; Refs. 6, 8, and
27), ()placing MANA in glycosyl hydrolase Family 26,
according to the classification of Henrissat and Bairoch(7) .
Inspection of this sequence reveals the highly conserved and unusual
protein motif of WFWWG. Upstream of this region (6 residues) is the
dipeptide sequence HE which is conserved in all of the enzymes in
Family 26 enzymes. Hydrophobic cluster analysis revealed two other
acidic residues at positions 371 and 376 that are conserved in the
Family 26 mannanases. (
)In view of the widespread
participation of glutamate and aspartate residues in the acid-base
catalysis of glycosidic bonds by
-glycanases, it is possible that
the conserved glutamates and aspartates in the four Family 26
mannanases are candidate nucleophiles or proton donors in the active
sites of these enzymes.
Figure 5:
Comparison of the primary structure of the
catalytic domain of Piromyces MANA with the corresponding
mannanases (MANA) of Bacillus and P. fluorescens subsp. cellulosa and an endoglucanase (EG) of Bacteroides ruminicola. The positions of the first and last
residues in the primary structures of the Bacillus mannanases(8, 27) , Pseudomonas MANA (6) , and the B. ruminicola EG are shown.
Those amino acids that exhibit identity or similarity in four of the
five sequences are boxed.
Derivatives of xynA, lacking only 48 bp of 3`-coding sequence (Fig. 1; pGX10) did not encode a functional xylanase. Insertion of the 5` 1539 bp of xynA into pMTL22p on a SstI/EcoRI restriction fragment, such that the ATG start codon was in-frame with the vector's lacZ`, generated a plasmid (pGX3) that also did not direct the synthesis of a functional xylanase. In contrast, cloning 1090 bp of the 3`-coding region of xynA into pMTL23p, such that the xylanase gene was in-frame with the vector's lacZ`, generated a plasmid (pGX7) that encoded a functional xylanase. These data suggest that while the C-terminal catalytic domain homologue exhibited catalytic activity, the putative N-terminal catalytic domain was inactive.
Figure 6: Docking of the reiterated 40-residue domain with components of the cellulose-binding cellulase-hemicellulase complexes from Neocallimastix and Piromyces. Cellulase-hemicellulase complexes from Piromyces (A) or N. patriciarum (B) were fractionated by SDS-PAGE (10% polyacrylamide gel) and stained with Coomassie Blue (lane 1) or electroblotted onto transfer membrane (lanes 2 and 3). Blots were probed with the GST-XYLA fusion protein (lane 2) or with GST alone (lane 3), and binding of the probe was revealed by treating with anti-GST antibodies as described under ``Materials and Methods.'' The sizes of protein standards and the polypeptide components that bound to the GST-XYLA fusion protein are shown in kilodaltons.
Figure 7:
Binding specificity of the conserved
duplicated 40 residue noncatalytic domain. In A, samples
containing 20 µg each of the cellulase complex from Piromyces sp. (lane 1), cell-free extract from E. coli JM83 (lane 2), and cellulosome from C. thermocellum (lane 3) were fractionated by SDS-PAGE, electroblotted
onto Immobilon-P membrane, and probed with the P-labeled
40-residue noncatalytic domain. The size of the hybridizing polypeptide
is shown in kilodaltons. In B, the same three samples (native,
nondenatured) were dot-blotted onto Immobilon-P at protein loadings of
10, 5, and 2.5 µg and probed with the duplicated domain. In C, cellulase complex from Piromyces sp. (20 µg)
was dot-blotted onto Immobilon-P and probed with radiolabeled probe
alone (lane 1) or with labeled probe that had been mixed 1:1 (lane 2), 1:5 (lane 3), or 1:10 (lane 4)
with unlabeled probe. The duplicated 40-residue noncatalytic fungal
domain was prepared and labeled with
P as described under
``Materials and Methods.'' Autoradiography was performed at
-70 °C for 24 h with intensifying
screens.
A primary objective of this study was to evaluate the extent
to which the noncatalytic reiterated 40-residue sequence is conserved
in anaerobic fungi and to investigate the function of this domain in
plant cell wall hydrolysis. To facilitate this objective, the primary
structures of two hemicellulases from the anaerobic fungus Piromyces were determined, and the relationship between the
structure and function of these two enzymes was investigated. Data
presented in this report indicate that XYLA contains two catalytic
domain homologues. Although other plant cell wall hydrolases comprising
of multiple catalytic domains, originating from distinct ancestral
genes, have been reported from C. saccharolyticum(5) and Ruminococcus
flavefaciens(29, 30) , the reiteration of
catalytic domains in cellulases and xylanases has only been observed
previously in enzymes derived from the anaerobic fungus Neocallimastix(12, 31) . The similarity in
the molecular organization of XYLA from Piromyces and xylanase
A from Neocallimastix supports the view that Neocallimastix and Piromyces are closely related or
that there has been extensive gene transfer between the two organisms.
In contrast to the Neocallimastix xylanase, which contained
duplicated catalytic domains that were both functional, only the
C-terminal domain of XYLA from Piromyces appears to exhibit
catalytic activity. However, this interpretation must be viewed with
some caution as: (i) it is possible that the folding of the protein in E. coli is different to that in the endogenous host, and the
consequence of misfolding is an inactive N-terminal domain; (ii) a
mutation in xynA, during the cloning of the cDNA, may have
resulted in the inactivation of the first catalytic region. This is
unlikely, because the same xylanase cDNA was isolated from a number of
distinct recombinant phage, and each form of the xylanase exhibited the
same properties. Inspection of the primary structure of the N-terminal
catalytic domain of the Piromyces xylanase did not provide an
explanation for its lack of catalytic activity; residues that are
conserved in the catalytic domains of all other Family 11 xylanases
were also present within XYLA. Of particular note is the conservation
of Glu and Glu
; these active site residues
function as the nucleophile and proton donor(32) ,
respectively, in the acid-base hydrolysis of glycosidic bonds mediated
by Family 11 xylanases. It is difficult, therefore, to evaluate
precisely why the N-terminal catalytic domain homologue is inactive.
The rationale for the evolution of a xylanase with a second, but
inactive catalytic domain is also not readily apparent. It is possible
that early in evolution xynA encoded an enzyme with two active
catalytic domains, and although a mutation occurred in the 5` region of
the gene resulting in the inactivation of the N-terminal domain, the
xylanase activity of the C-terminal region of XYLA was sufficient
selective pressure to ensure the retention of xynA within the Piromyces genome.
This paper describes, for the first time,
the primary structure and molecular architecture of a mannanase from an
anaerobic fungus. The location of the fungal enzyme in a glycosyl
hydrolase Family (Family 26) that also contains bacterial mannanases,
provides evidence for a common evolutionary origin for the rumen fungal
and bacterial mannanases, and raises the possibility that Piromyces
manA arose through the lateral transfer of a prokaryotic gene to
the eukaryotic microorganism. Inspection of the enzymes belonging to
Family 26 reveals both mannanases and endoglucanases, suggesting that
these two enzyme species evolved from a common progenitor sequence.
Whether the ancestral sequence was an endoglucanase, mannanase, or
-glycanase with a broad substrate specificity remains to be
elucidated. However, it is interesting to note that, while some
endoglucanases display xylanase activity(33, 34) , the
endoglucanases and mannanases in Family 26 exhibit no detectable
cross-specificity, even though the two enzyme species have evolved from
the same ancestral sequences(7) .
A further major objective of this study was to evaluate the function of the highly conserved noncatalytic 40-residue sequence located in anaerobic fungal cellulases and hemicellulases. Noncatalytic repeated sequences have been observed in a number of cellulases and xylanases (3, 35) , and the role of these reiterated sequences has been defined for certain enzymes. For example, duplicated noncatalytic CBDs have been identified in cellulases from C. saccharolyticum, Cellulomonas fimi, Clostridium cellulolyticum, and Clostridium stercorarium(3) . In addition, the duplicated 95-residue sequences, which exhibited 65% sequence identity, and are located in xylanase D from C. fimi, constitute a CBD and noncatalytic xylan-binding domain, respectively(36, 37) . In addition to repeated CBDs, a 23-residue duplicated sequence has been observed in cellulases and xylanases from C. thermocellum and C. cellulolyticum(3) . This sequence plays an important role in the docking of the cellulases and xylanases to a noncatalytic scaffolding protein to form a multienzyme cellulase complex referred to as the ``cellulosome''(28, 38) . Data presented in this study clearly showed that the reiterated noncatalytic domain observed in anaerobic fungal plant cell wall hydrolases is not a polysaccharide binding domain, but functions as a protein docking sequence that interacts with polypeptides of 116 and 97 kDa that are present in the multienzyme cellulase-hemicellulase complexes of Neocallimastix and Piromyces, respectively. Although the role of this protein docking domain has not been defined, we suggest that the polypeptides to which the conserved noncatalytic domain binds are the scaffolding proteins and that this protein-protein interaction mediates the assembly of the multienzyme cellulase-hemicellulase complexes synthesized by these eukaryotic microorganisms. It should be emphasized, however, that other mechanisms could also contribute to the formation of the eukaryotic enzyme complexes. For example, it is possible that, in addition to the binding of the protein docking sequence to a scaffolding protein, direct interactions between the catalytic domains could also exist.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X91857 [GenBank]and X91858[GenBank].