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Journal of Bacteriology, September 1999, p. 5288-5295, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cloning and Sequence Analysis of a New Cellulase
Gene Encoding CelK, a Major Cellulosome Component of Clostridium
thermocellum: Evidence for Gene Duplication and
Recombination
Irina
Kataeva,1
Xin-Liang
Li,1
Huizhong
Chen,1
Sang-Ki
Choi,2 and
Lars G.
Ljungdahl1,*
Center for Biological Resource Recovery and
Department of Biochemistry & Molecular Biology, The University of
Georgia, Athens, Georgia 30602-7229,1 and
Laboratory of Molecular Genetics, National Institute of Child
Health and Human Development, National Institutes of Health,
Bethesda, Maryland 20892-27852
Received 26 February 1999/Accepted 22 June 1999
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ABSTRACT |
The cellulolytic and hemicellulolytic complex of Clostridium
thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding
polypeptide. We report here a new cellulase gene, celK,
coding for CelK, a 98-kDa major component of the cellulosome. The gene
has an open reading frame (ORF) of 2,685 nucleotides coding for a
polypeptide of 895 amino acid residues with a calculated mass of
100,552 Da. A signal peptide of 27 amino acid residues is cut off
during secretion, resulting in a mature enzyme of 97,572 Da. The
nucleotide sequence is highly similar to that of cbhA
(V. V. Zverlov et al., J. Bacteriol. 180:3091-3099, 1998), having
an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the
same bacterium. Homologous regions of the two genes are 86.5 and 84.3%
identical without deletion or insertion on the nucleotide and amino
acid levels, respectively. Both have domain structures consisting of a
signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a
330-amino-acid insertion in CbhA between the catalytic domain and the
dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size
difference between CelK and CbhA. Upstream and downstream flanking
sequences of the two genes show no homology. The data indicate that
celK and cbhA in the genome of C. thermocellum have evolved through gene duplication and
recombination of domain coding sequences. celK without a
dockerin domain was expressed in Escherichia coli and
purified. The enzyme had pH and temperature optima at 6.0 and 65°C,
respectively. It hydrolyzed
p-nitrophenyl-
-D-cellobioside with a
Km and a Vmax of 1.67 µM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of
CelK activity, with a Ki of 0.29 mM. The enzyme
was thermostable, after 200 h of incubation at 60°C, 97% of the
original activity remained. Properties of the enzyme indicated that it
is a cellobiohydrolase.
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INTRODUCTION |
Clostridium thermocellum
secretes into the cultural medium a multiprotein complex, termed
cellulosome, capable of efficient hydrolysis of highly ordered
crystalline cellulose (3, 15). It contains 14 to 26 different polypeptides and possesses endo- and exoglucanase, xylanase,
mannanase, lichenase, and feruloyl esterase activities (8, 23,
36). All cellulosomal components have modular structures (5,
38). The enzymatically active components are composed of at least
a catalytic domain and a highly conservative type I dockerin domain
(5). Some of the enzymes are more complex and include
cellulose binding domains (CBD), S-layer-homologous domains, and
domains of unknown functions (38). The largest cellulosome
subunit is a 210-kDa enzymatically inactive scaffolding protein, CipA
(17). It is composed of nine highly similar cohesin domains
interacting with dockerin domains of catalytic subunits,
(42), a family III CBD binding the cellulosome to the
cellulose, and a special dockerin type II domain attaching the complex
to the cell surface (29). A high degree of homology between
CipA cohesin domains (17) together with studies on the interactions between different cohesin domains and some catalytic subunits suggest that binding of the catalytic subunits to CipA occurs
on random basis (21, 27, 32). This seems to indicate that
the incorporation of a specific catalytic subunit into the cellulosome
depends on its relative amount and that predominant enzymes play
important roles in the cellulosome.
Many genes encoding cellulosomal components have been cloned, and their
products have been characterized (3, 5, 15). Surprisingly, a
98-kDa protein, the presence of which, in relatively large amounts, in
the cellulosome was described by Choi and Ljungdahl (10),
has been neither sequenced nor characterized.
This report describes in detail the cloning and sequencing of
celK. CelK, the product of celK, has a high
degree of homology with CbhA (51), a 138-kDa
cellobiohydrolase from the same bacterium. CelK expressed in
Escherichia coli was purified, and its enzymatic properties
indicate strongly that it is a cellobiohydrolase. Thus, the cellulosome
of C. thermocellum contains at least three
cellobiohydrolases, CelS, CbhA, and CelK. (A preliminary report
covering some properties of CelK was given at the MIE BIOFORUM 98 conference on the Genetics, Biochemistry and Ecology of Cellulose
Degradation [22].)
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MATERIALS AND METHODS |
Bacterial strains, culture conditions, and plasmids.
C.
thermocellum JW20, described by Freier et al. (16), was
used for isolation of genomic DNA and cellulosomes. Culture conditions were as described by Wiegel (49); 1% (wt/vol) cellobiose
and 5% (wt/vol) Avicel PH-101 were used as carbon sources. E. coli INVaF' (Invitrogen Inc., Carlsbad, Calif.) and JM109
(Stratagene Cloning Systems, La Jolla, Calif.), used as cloning hosts
for pCR2.1 (Invitrogen) and pBluescript SK(+) (Stratagene),
respectively, were grown in Luria-Bertani medium supplemented with
ampicillin (100 µg/ml).
Isolation and internal peptide sequencing of CelK.
Cellulosomes (100 µg) purified from 3-day-old-culture as described
earlier (10) were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (26).
The concentration of acrylamide was 7.5% (wt/vol). After
electrophoresis, the proteins were transferred to a polyvinylidene
difluoride membrane and stained with Ponceau S dye. The CelK band
(~98 kDa) was identified according to the banding pattern of
cellulosomal proteins on gels used for SDS-PAGE (10),
excised with a razor blade, rinsed with 0.5 ml of distilled water, and
then digested with protease Lys-C as specified by the supplier
(Boehringer Mannheim, Indianapolis, Ind.). Residual peptides were
separated by means of Hewlett-Packard (Wilmington, Del.) 1100 series
high-pressure liquid chromatography (HPLC) control module equipped with
a V8 reverse-phase column. Peptide peaks were monitored by UV
absorption at 280 nm. N-terminal amino acid sequences of selected
peptides were determined by using an Applied Biosystems model 477A
gas-phase sequencer with an automatic on-line phenylthiohydantion analyzer.
Isolation of genomic DNA from C. thermocellum.
Genomic
DNA was purified from a 0.5-liter culture by the method of Marmur
(33), with the following modifications. After treatment of
the cells with lysozyme (5 mg/ml) for 4 h, SDS (0.1%, wt/vol) and
proteinase K (500 µg/ml) were added. The solution was incubated at
37°C and then dialyzed for 24 h against 1 liter of 100 mM
Tris-HCl buffer (pH 7.5) containing 10 mM EDTA and 150 mM NaCl. The
dialysate was incubated with DNase-free RNase (50 µg/ml) for 30 min
at 37°C. Genomic DNA was extracted repeatedly (30 min for each
extraction) with phenol-chloroform-isoamyl alcohol (12:12:1) at 37°C.
DNA from the upper phase was precipitated with 0.1 volume of 3 M of
sodium acetate (pH 5.2) and 2.5 volume of cold ethanol. After
incubation at 
20°C for 30 min, DNA was collected by centrifugation
(7,000 × g, 30 min) and then carefully resuspended in
2.5 ml of distilled water. The suspension was then incubated at 37°C
overnight for slow hydration. Purity and size of the genomic DNA were
determined on the basis of absorption at 260 and 280 nm and by 1%
agarose gel electrophoresis in the presence of ethidium bromide.
Primer design, PCR, and cloning.
Degenerate oligonucleotides
were designed according to protein (peptide) sequences (Table
1) and synthesized with an Applied Biosystems DNA synthesizer. Using the oligonucleotides in combination as primers and purified genomic DNA as a template, PCRs were done on a
model 480 thermal cycler (Perkin-Elmer, Norwalk, Conn.). All reagents
were purchased from Perkin-Elmer and used as instructed. Annealing
temperatures were 42 and 54°C with degenerate and specific primers,
respectively; extension time was from 1 to 4 min, depending on the
length of amplified fragments. PCR products (10 µl) were analyzed on
agarose gels in the presence of ethidium bromide. They were either
sequenced directly or cloned into the pCR2.1 vector with a TA cloning
kit (Invitrogen) and then sequenced (see below).
DNA sequencing and sequence analysis.
PCR products were
purified by using either Microcon tubes (Amicon, Beverly, Mass.) or a
Geneclean Spin kit (Bio101) before subjected to sequencing. Plasmids
were purified from overnight-grown E. coli cultures by using
a QIAprep Spin Plasmid Miniprep kit (Qiagen, Valencia, Calif.). DNA
fragments were sequenced, using universal and sequence specific primers
by means of an automatic PCR sequencer (Applied Biosystems). The
Genetics Computer Group (GCG) program (version 8; GCG, University of
Wisconsin Biotechnology Center, Madison) on the VAX/VMS system of the
BioScience Computing Resource at the University of Georgia was used to
analyze sequence data.
Southern blot analysis and colony hybridization.
Genomic DNA
of C. thermocellum was digested with a single restriction
enzyme and with enzymes in combinations. The digested fragments were
separated by electrophoresis and transferred to a nylon membrane with a
Turboblotter (Schleicher & Schuell, Keene, N.H.). Transfer of colonies
to nylon membranes was done as described by Sambrook et al.
(39). DNA was cross-linked to the membrane with a UV
cross-linker (Stratagene). Hybridization probes for DNA were generated
by PCR amplification in the presence of digoxigenin-labeled dUTP
(Boehringer Mannheim). Hybridization with the labeled probe, stringency
washing, and detection of positive bands or colonies were done as
instructed by Boehringer Mannheim.
CelK purification.
A 10-liter culture of E. coli
BL21(DE3)(pLys) harboring pET-21b(+) containing celK lacking
the dockerin domain coding region (nucleotide residues 3594 to 3829)
was grown to an optical density at 600 nm of approximately 0.8. After
addition of 2 mM isopropyl--D-thiogalactopyranoside, the
culture was incubated at 37°C for 5 h. The cells were collected, washed with 50 mM sodium phosphate buffer (pH 7.8), and broken in a
French press. Cell debris was removed by centrifugation. The clear
supernatant was mixed at 4°C with an increasing amount of
Ni-nitrilotriacetic acid resin (Xpress protein purification System;
Invitrogen) until the activity of CelK in the supernatant was
negligible. The suspension was packed into a column. Washing and
elution conditions were as recommended by the supplier. Fractions containing p-nitrophenyl--D-cellobioside
(PNP-cellobioside) activity were combined and dialyzed against 20 mM
Tris-HCl buffer, pH 7.5. The dialyzate was applied to a Mono Q HR10/10
column, and proteins were eluted with a gradient from 0 to 0.6 M NaCl.
Fractions containing CelK were concentrated and chromatographed on a
Superose 12 HR10/30 column in the presence of 0.1 M NaCl.
Enzyme assay and analytical methods.
The activity of CelK
was assayed at 65°C in 50 mM sodium citrate buffer, pH 6.0. The
concentration of PNP-cellobioside was 5 mM. Hydrolysis of
PNP-cellobioside was determined by the release of
p-nitrophenol. One enzyme unit was defined as amount of the enzyme releasing 1 µmol of p-nitrophenol per min. To test
the dependence of CelK activity on pH, the following buffers (50 mM each) were used: histidine-HCl (pH 5.0 to 6.0), sodium phosphate (pH
6.0 to 7.0), and Tris-HCl (pH 7.0 to 9.0). The
Km for PNP-cellobioside and
Ki for cellobiose were determined at 65°C in
50 mM sodium citrate buffer (pH 6.0).
Nucleotide sequence accession number.
The nucleotide
sequence of celK of C. thermocellum JW20 has been
assigned accession no. AF039030 in the GenBank database.
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RESULTS |
Both celK and cbhA are present in the
C. thermocellum genome.
One of the most abundant
subunits of the C. thermocellum cellulosome (here designated
CelK) has a mass of 98 kDa with an N-terminal amino acid sequence of
LEDKSSKLPDYKNDLLYE (10).
Database search revealed that of the 19 amino acid residues, there were
three mismatches (in boldface) with that of the N-terminal region of
CbhA (formerly Cbh3; accession no. X80993), which contains 1,230 amino
acid residues with molecular mass of 138,007 Da. To clarify the
relationship between the 98-kDa subunit and CbhA amino acid sequences,
purified CelK was digested with Lys-C protease. Two internal peptides,
EYYFK and IPIEMPYAGGEQ, were obtained after separation by HPLC. The
first short sequence was not found in the deduced protein sequence of
CbhA, although two YYF sites (residues 284 to 286 and 1044 to 1046)
were identified. The second internal sequence matched perfectly with
amino acid residues of 326 to 337 of CbhA. The differences with respect
to size and partial amino acid sequence indicated that the 98-kDa component and CbhA may be encoded by two distinct genes of C. thermocellum.
Degenerate oligonucleotides CelK1F and CelK1R (Table
1), corresponding
to the less identical sites of the 98-kDa N-terminal
sequence and the
longer internal peptide, were used to amplify
the putative gene coding
for the 98-kDa protein. A 0.9-kb DNA
fragment was obtained (data not
shown) after amplification. This
size is in agreement with the
corresponding region of CbhA. Southern
blotting using the amplified
0.9-kb fragment as a hybridization
probe should detect at least two
signals, assuming that the two
genes
celK and
cbhA have high level of homology (Fig.
1). Indeed,
two
signals were obtained with genomic DNA digested with
EcoRI
(lane 1),
DraI (lane 2), or
BamHI (lane 6) under
low-stringency
washing (A). As the stringency washing temperature was
increased
from 42°C (A) to 55°C (B) and 65°C (C), one of the two
signals
became less intense or disappeared (e.g., 3.0-, 3.9-, and
3.0-kb
bands with
EcoRI,
DraI, and
BamHI, respectively [Fig.
1B and
C]).
The sizes of these bands matched
those found in the sequenced
cbhA. The results strongly
suggest the presence of two separate
genes encoding
CelK and
CbhA in the genome of
C. thermocellum.
Only one
signal was found with genomic DNA digested with
ApaI
(lane
3),
NotI (lane 4), and
AscI (lane 5) at the three
washing
temperatures, and all of these bands were larger than 21.2 kb.
This means that there are no restriction sites involving these
enzymes
in the DNA regions flanking the
celK and
cbhA
genes or,
alternatively, that these enzymes failed to digest the DNA
sample
efficiently.
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FIG. 1.
Southern analysis of C. thermocellum genomic
DNA. Genomic DNA was digested with EcoRI (lane 1),
DraI (lane 2), ApaI (lane 3), NotI
(lane 4), AscI (lane 5), and BamHI (lane 6) and
fractionated on an agarose gel (1%). DNA fragments in the gel were
transferred to a nylon membrane. The hybridization probe was the 0.9-kb
PCR fragment amplified by using the CelK1F and CelK1R (Table 1).
Digoxigenin was incorporated into the fragment during PCR amplification
in the presence of digoxigenin-conjugated dUTP (Boehringer Mannheim).
Stringency washing was done twice for 15 min at 42°C (A), 55°C (B),
and 65°C (C). DNA standards labeled with digoxigenin were run under
identical conditions, and their positions of migration are shown on the
left.
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The 0.9-kb PCR product was cloned into pCR2.1, and its nucleotide
sequence was determined. Sequence analysis demonstrated
that this
fragment was different from but highly similar to the
5' half of the
cbhA open reading frame (ORF). Identity on the
nucleotide
level between the two ORFs was 74.2%. The deduced amino
acid sequence
of CelK revealed 74.1% identity with that of CbhA
without any deletion
or insertion. Furthermore, the amino acid
sequences of the N terminus
and the two internal peptides of the
98-kDa protein all matched those
of the deduced amino acid sequence
of the PCR product except for one
residue: EYYFK was obtained
by protein sequencing, whereas GYYFK was
obtained by deduction.
This mismatch may be attributable to
experimental error during
the protein sequencing or PCR cloning.
Combined, the data clearly
show that two highly similar genes are
present in the genome of
C. thermocellum. We propose the
name
celK for the gene coding
for the 98-kDa cellulosomal
subunit, and CelK for its encoded
polypeptide, consistent with the
nomenclature for the recently
published cellulase gene,
celJ
(
1).
Several steps were taken to obtain the complete nucleotide sequence of
celK and its upstream and downstream regions. First,
we took
advantage of the fact that CelK is a subunit of the cellulosome
and
therefore its dockerin domain, particularly the first peptide
of that
domain, should be highly similar to that of many other
subunits
(
10). A 1.7-kb DNA fragment was amplified by PCR using
a
pair of primers, CelK2F (with low homology to the corresponding
site of
cbhA) and CelK2R (a degenerate primer corresponding to
the
first conserved region of the dockerin domain) (Table
1).
The
nucleotide sequence of the PCR fragment allowed us to generate
two
hybridization probes corresponding to the 5' and 3' halves
of
celK and then obtain two separate plasmid clones spanning
the
complete ORF plus 1.1-kb 5' and 0.6-kb 3' ends. Therefore, the
nucleotide sequence determined included a total of 4.187 kb of
the
celK locus. A restriction map of
celK is shown in
Fig.
2A.
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FIG. 2.
(A) Restriction map of celK. D, E, H, and X,
restriction sites for DraI, EcoRI,
HindIII, and XhoI, respectively. (B and C)
Comparisons between partial sequences of celK and
cbhA. Sequences compared include 5' (B) and 3' (C) ends of
the two ORFs, together with some untranslated regions. Numbers on the
left correspond to those in the databases. The deduced amino acid
sequence for celK is shown as boldface; putative ribosome
binding and transcription terminator sites are underlined.
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To verify the presence of the two highly similar genes encoding CelK
and CbhA in the strain of
C. thermocellum JW20 used for
genomic DNA extraction, we amplified the regions of the 3'-terminal
halves of the two genes where
cbhA has about a 900-bp
addition
in comparison with
celK (see below). Two bands with
sizes of about
650 and 1,600 bp (Fig.
3)
were detected after PCR amplification.
These two bands matched the
sizes of the corresponding regions
of
celK and
cbhA, respectively, supporting the existence of both
genes
in the genome of the
C. thermocellum JW20. The band of
celK was more intensive than the one corresponding to
cbhA, probably
because the reverse primer was less
homologous to the sequence
of
cbhA and/or the PCR conditions
favored the amplifications of
shorter products (
celK). The
presence of two bands, 150 and 98
kDa, recognized with anti-CbhA
antibodies in the cellulosome indicates
that
C. thermocellum
F7, a Russian isolate, also contains the
two highly homologous genes
(
51).
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FIG. 3.
Analysis of PCR products amplified from C. thermocellum genomic DNA, using CelK4F and Celk2R (Table 1) as
primers. The gel was loaded with PCR products amplified in the presence
(lane 1) and absence (lane 2) of template. Lane 3 was loaded with 1-kb
DNA standards (Promega).
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Characterization of celK and CelK.
Sequence
analysis revealed that celK had an ORF of 2,685 nucleotides.
The G+C content of the ORF was 43.3%, characteristic of C. thermocellum genes sequenced to date (5, 27). The start codon was determined based on the fact that there is a stop codon preceding the ATG codon and that the signal peptide had all of the
properties found for signal peptides of bacterial extracellular enzymes (48). A putative ribosome binding site (GGAGG)
was found 8 bp before the ATG codon. Upstream of the coding
region are possible promoter sequences, TTGATG for the 35
region and TAATTT for the 10 region. A region of 20-bp
palindrome sequences downstream of the TAA stop codon might serve as a
transcription terminator. Analysis of 1,142-bp upstream and 356-bp
downstream sequences failed to detect any ORF. These data suggest that
celK, like most genes coding for hydrolytic enzymes of
C. thermocellum, is monocistronic (4).
The deduced CelK contained 895 amino acid residues with a molecular
mass of 100,713 Da. Residues 28 to 45 matched perfectly
the N-terminal
amino acid sequence determined for the 98-kDa subunit
of the
cellulosomes (
10). Thus, residues 1 to 27 of CelK served
as
the signal peptide, and the calculated mass 97,572 Da of the
mature
CelK was consistent with that determined by SDS-PAGE (
10),
indicating that glycosylation of the 98-kDa polypeptide was, if
present, negligible. The low degree of glycosylation of the CelK
could
be explained by the absence of long P/T-rich linkers characteristic
for
CipA and shown to be highly glycosylated (
18,
27). The
agreement of amino acid sequences determined by protein sequencing
and
by deduction from nucleotide sequence for CelK confirmed the
differences between CelK and CbhA on the nucleotide level and
therefore
excluded the possibility that the 98-kDa subunit revealed
by SDS-PAGE
was a proteolytic product of
CbhA.
Homology studies.
The complete amino acid sequences of CelK
and CbhA were compared by using the GCG program BESTFIT (Fig.
4). Very high sequence identity (83.4%)
and similarity (90.5%) between their homologous regions were observed
with the addition of only one amino acid residue (position 687) in
CbhA. Identity on the nucleotide level between the homologous regions
was 86.5%. The 5' and 3' untranslated sequences of the genes revealed
substantially lower sequence identity (Fig. 2B and C, respectively). A
328-amino-acid-residue region corresponding to residues 815 to 1143 of
CbhA was absent in CelK. This region in CbhA explains the size
difference (98 versus 130 kDa) between the two proteins.
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FIG. 4.
Comparison between the deduced amino acid sequences of
celK and cbhA. The sequence in CbhA containing a
family III CBD is underlined. The signal peptide in CelK is in
boldface; amino acids in CelK determined by protein sequencing are
italicized.
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In a recent publication (
51), the multidomain structure of
CbhA was reported. Analysis of the amino acid sequence of CelK
deduced
from its nucleotide sequence revealed a domain structure
of the
polypeptide similar to that of CbhA (Fig.
5). An N-terminal
signal peptide was
followed by a fragment of 160 amino acids (positions
40 to 200) that
showed some homology to family IV bacterial CBDs
(Fig.
6). It displayed sequence identities of
77% to the CBD of
C. thermocellum CbhA (
51),
30% to the CBD N1 of
Cellulomonas fimi CenC
(
11), 28% to the CBD I of
C. thermocellum LicA
(direct
submission; accession no.
X89732), 27% to the CBD of
Streptomyces reticuli Cel1 (
40), 22% to the CBD
of
Thermomonospora fusca E1 (
28), 25% to the CBD
of
Thermotoga neapolitana LamA (
12),
24% to the
CBD N2 of
C. fimi CenC (
11), and 24% to the CBD
II
of
C. thermocellum LicA.
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FIG. 5.
Domain organizations of C. thermocellum CelK
and CbhA. Assignments of domains are based on sequence similarities to
domains of known functions. Symbols: SP, signal peptide; CBD IV, CBD of
family IV; CD, catalytic domain; DD, dockerin domain; CBD III, CBD of
family III; Fn3, fibronectin type 3-like domain.
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FIG. 6.
Alignment of the CBD of CelK with CBDs of family IV
found in microbial cellulases. Abbreviations: Celk_Clotm, C. thermocellum CelK; Cbha_Clotm, C. thermocellum CbhA;
Cenc_Celfi, Cellulomonas fimi CenC; Lica_Clotm, C. thermocellum LicA; Cel1_Strre, S. reticuli Cel1;
E1_Thfu, T. fusca E1; Lama_Thne, Thermotoga
neapolitana LamA.
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The catalytic domain of CelK was next to the CBD of family IV. The
amino acid sequence up to about residue 600 was homologous
to a number
of cellulases (Fig.
7) belonging to
glycosyl hydrolase
family 9 (
38). This region of CelK
displayed sequence identities
of 90% to
C. thermocellum
CbhA (
51), 47% to
Cellulomonas fimi CenC
(
11), 46.5% to
S. reticuli CelA (
40),
44.5% to
Pseudomonas fluorescens CelA (
19), and
31.1% to
Butyrivibrio fibrosolvens Ced1 (
6). All
those except CelK and CbhA listed in Fig.
7 have
been demonstrated to
be endoglucanases with inverting mechanisms
(
38), whereas
CbhA expressed in
E. coli has been shown to be
a
cellobiohydrolase (
41). It is not clear why CbhA and CelK
(see below) from
C. thermocellum cellulosome differ in
enzymatic
properties from the other homologous cellulolytic enzymes.
Clearly,
the sequence differences are sufficient to distinguish their
catalytic
modes. This is the case between fungal type II
cellobiohydrolases
and bacterial endoglucanases all belonging to
glycosyl hydrolase
family 6 (
38). This hypothesis may be
confirmed by performing
deletions from the ends of CbhA or CelK when
expressed in
E. coli and/or by comparison of
three-dimensional structures of the two
types of enzymes. CelK as well
as CbhA displayed only weak homology
to CelS, a well-established
cellobiohydrolase in the
C. thermocellum cellulosome
(
24,
25,
47), suggesting that CelK and CbhA
represent a
second type of cellobiohydrolase in the cellulosome.
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FIG. 7.
Alignment of the catalytic domain of CelK with those of
other cellulases. Amino acid sequences that showed over 30% identity
to the entire catalytic domain of CelK include CbhA of the same
organism (CbhA_Clotm), CenC of Cellulomonas fimi
(CenC_Celfi), CelA of S. reticuli (Cela_Strre), and CelA of
B. fibrosolvens (Cela_Butfi).
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Located on the C terminus of CelK is the dockerin domain found in a
number of enzymes of the
C. thermocellum cellulosome.
This
domain allows the catalytic subunits to interact with the
cohesins of
CipA, the scaffolding protein of the complex (
42).
Thus, all
available evidence indicates that CelK is a component
of the
cellulosomal
complex.
A summary of the putative domain composition of CelK and CbhA presented
in Fig.
5 demonstrates that the 328-amino-acid fragment
of CbhA located
between its catalytic and dockerin domains, missing
in CelK, is
composed of a Fn3 domain and a CBD of family
III.
Properties of CelK.
CelK devoid of the dockerin domain
expressed in E. coli has a molecular mass of 94 kDa (Fig.
8), which is in a good agreement with
deduced molecular mass of the enzyme. This truncated CelK had a
temperature optimum of 65°C and an optimum pH of 6.0. The enzyme was
thermostable; after 200 h of incubation at 60°C, 97% of the
original activity remained. CelK did not hydrolyze xylan, glucomannan,
or cellobiose. It was active toward PNPC, with a Km and Vmax of 1.67 µM
and 15.1 U/mg, respectively. Cellobiose was an efficient inhibitor of
CelK, with a Ki of 0.29 mM. We have demonstrated
that CelK did not decrease the viscosity of carboxymethylcellulose and
that it hydrolyzed carboxymethylcellulose to cellobiose, cellotriose to
glucose and cellobiose, cellotetraose to cellobiose, and cellopentaose to glucose and cellobiose (22). All of these results
strongly indicate that CelK is a cellobiohydrolase.
| |
DISCUSSION |
Two genes coding for two cellulosomal subunits, CelK and CbhA,
with highly similar catalytic domains are present in the genome of
C. thermocellum. The regions of the catalytic domains have identities of about 90% on both nucleotide and amino acid levels, a
level of homology that has not been found between any two genes of
C. thermocellum sequenced to date (5). Identities
between cellulases or between xylanases of C. thermocellum
have never exceeded 50%. We propose that catalytic sites of
celK and cbhA arose from a common ancestral gene
by duplication. Duplication of genes coding for cellulases and other
hydrolases is more common for anaerobic fungi (31). Thus,
highly similar mannanases (14, 35) and cellulases (9,
30) are present in the same species of the anaerobic fungi;
examples are the several multiple cellulases of the anaerobic fungus
Orpinomyces strain PC-2, which contain highly similar
catalytic domains. Gene duplication has been found to be common in
organisms from bacteria to mammals and believed to be critical for
evolution but appears unusual for genes of C. thermocellum.
The fact that the catalytic domains of CelK and CbhA were duplicated
and then favorably selected suggests that these catalytic domains may
be essential for the bacterium to engage in cellulose degradation. In
addition to the catalytic domains, CelK and CbhA have very similar
dockerin domains. The striking distinction between the enzymes is the
328-amino-acid region located close to the C terminus. This region,
which contains a combination of fibronectin type 3-like domain and a
CBD of family III (51), is absent in CelK. The domain
organization shown in Fig. 4 implies that sequences coding for
different domains of the cellulosomal subunits evolved independently
and then combined to code for the complete polypeptides. If this is
true, then possibly celK was formed by combining regions of
the family IV CBD and catalytic domain from one side with the dockerin
domain from another and then was inserted by the sequences coding for
the fibronectin-like domain and the family III CBD to yield
cbhA. Alternatively, cbhA was first present and
then lost the fragment described to become celK.
Nevertheless, duplication and rearrangement involving sequences encoding separate domains were needed to yield the two complete genes.
CBDs are believed to be important in increasing local concentration of
the catalytic domains to the substrate and/or in disrupting the
hydrogen bonds between cellulose chains. These domains play important
roles in free-acting enzymes (20, 43, 46). The role of CBDs
found in some cellulosomal catalytic components is not yet clear. The
cellulosomal enzymes are attached to the cellulose surface by means of
CipA containing a CBD of family III and in fact do not need their own
CBDs. However, CBDs are found in several cellulosomal cellulolytic
subunits, including CelF (containing a CBD of family III)
(44), CelE (CBD of family VII) (38), CbhA (two
CBDs of families III and IV) (51), and CelK (CBD of family
IV). Another subunit of the cellulosome, XynZ, contains a CBD of family
VI (44). It has been demonstrated that the family III CBD of
CipA binds to both amorphous and crystalline cellulose, but its binding
capacity with amorphous cellulose is 20 times higher (37).
CelK CBD binds efficiently to acid-swollen cellulose and weakly to
Avicel (22). Properties of CBDs of the catalytic cellulosome
components have not been studied. These domains belong to at least four
different families, and a particular type of CBD is usually associated
with a particular type of catalytic domain (38, 44): CBDs of
family III of CelF and CbhA as well as CBDs of family IV of CelK and
CbhA with family 9 catalytic domains; CBD of family VII of CelE with a
family 5 catalytic domain; and CBD of family VI of XynZ with a family
10 catalytic domain (38). The presence of different CBDs in
several cellulosomal enzymes suggests that these domains play
significant and specific roles in cellulose degradation. The chemical
simplicity of cellulose belies its structural complexity. The diversity
of CBDs found in cellulosomal subunits may be necessary for binding of
the complex to various regions of cellulose regardless of the degree of
its crystallinity and other peculiarities of its structure. Perhaps CBDs are in some way involved in the hydrolysis process
(13).
For a long time it has been believed that the cellulosome contains
mostly endoglucanases (34). The discoveries of CelS
(24), CbhA (51), and, as reported here, CelK
indicate that cellobiohydrolases play an important role in cellulose
degradation by the cellulosome of C. thermocellum. This idea
is further supported by the fact that CelS and CelK are the most
abundant components of the cellulosome. The synergism between
cellulases in terms of cellulose hydrolysis is not limited to between
cellobiohydrolases and endoglucanases but also occurs between two
classes of cellobiohydrolases, one cleaving cellobiose from the
reducing ends and the other doing so from the nonreducing ends of
cellulose chains (2). High homology observed between CelK
and CbhA together with lack of significant homology between these
enzymes and CelS may reflect the presence of two different types of
cellobiohydrolases in the cellulosome.
| |
ACKNOWLEDGMENTS |
This work was funded by grant DE-FG02-93ER20127 from the
Department of Energy. Support by a Georgia Power Distinguished
Professorship in Biotechnology (to L.G.L.) is also gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, A214 Life Sciences Building, The University of Georgia, Athens, GA 30602-7229. Phone: (706)-542-7640. Fax: (706)-542-2222. E-mail: larsljd{at}arches.uga.edu.
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Abstract
Introduction
