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THE

CHEMICAL
Studies of Thermobifida fusca Plant Cell
RECORD Wall Degrading Enzymes

DAVID B. WILSON
Department of Molecular Biology & Genetics, Cornell University, 458 Biotechnology Building,
Ithaca, NY 14853, USA

SYNOPSIS: I have been studying the Thermobifida fusca cellulose degrading proteins for the past
25 years. In this period, we have purified and characterized the six extracellular cellulases and an
intracellular b- glucosidase used by T. fusca for cellulose degradation, cloned and sequenced the struc-
tural genes encoding these enzymes, and helped to determine the 3-dimensional structures of two
of the cellulase catalytic domains. This research determined the mechanism of a novel class of cel-
lulase, family 9 processive endoglucanases, and helped to show that there were two types of exocel-
lulases, ones that attacked the non-reducing ends of cellulose and ones that attacked the reducing
ends. It also led to the sequencing of the T. fusca genome by the DOE Joint Genome Institute. We
have studied the mechanisms that regulate T. fusca cellulases and have shown that cellobiose is the
inducer and that cellulase synthesis is repressed by any good carbon source. A regulatory protein
(CelR) that functions in the induction control has been purified, characterized, and its structural
gene cloned and expressed in E. coli. I have also carried out research on two rumen bacteria, Pre-
votella ruminicola and Fibrobacter succinogenes, in collaboration with Professor James Russell, helping
to arrange for the genomes of these two organisms to be sequenced by TIGR, funded by a USDA
grant to the North American Consortium for Genomics of Fibrolytic Ruminal Biology. © 2004 The
Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 4: 72–82; 2004: Published
online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20002

Key words: Thermobifida fusca; cellulose binding domain; endocellulase; exocellulase; xylanase;
xyloglucanase; Fibrobacter succinogenes; Prevotella ruminicola; protein engineering; regulation;
site directed mutagenesis; structure; synergism.

I first started studying microbial cellulases in 1982, when T. fusca YX produced a very active extracellular protease
Dr. Dexter Bellamy came to Cornell from General Electric that created many proteolytic artifacts. This enzyme3 was puri-
and brought a set of thermophilic cellulolytic bacteria, which fied, characterized, and its structural gene was cloned and
he had isolated1. On his recommendation, I chose to study sequenced4. This enzyme resembles a lytic protease in having
Thermobifida fusca YX (then called Thermomonospora fusca) an essential chaperon in an N-terminal pre-sequence and the
and set out to purify and characterize the set of extracellular entire amino acid sequence shows 46% identity to the a lytic
cellulases that it produced. T. fusca (Fig. 1) is a filamentous soil protease precurser5. This enzyme has high activity on most pro-
bacteria that grows at 55°C in defined medium on cellulose as
a carbon source. It appears to be a major degrader of plant cell
walls in heated organic material such as compost piles and  Correspondence to: Professor David B. Wilson; Phone: 607-255-5706;
rotting hay2. Fax: 607-255-6249; Email: dbw3@cornell.edu

The Chemical Record, Vol. 4, 72–82 (2004)


72 © 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.
Microbial Cellulases

sive studies of binding and synergism by T. fusca and T. reesei


cellulases and their catalytic and binding domains13–19. He also
developed a method for determining the rate of fragmentation
of Avicel particles using a particle counter20 and used it to show
that fragmentation is linear with time and enzyme concentra-
tion unlike the rate of reducing sugar production, which is
nonlinear with both time and enzyme concentration. He also
studied the effect of particle size on fragmentation21.
We also cloned and sequenced four endocellulase genes
from T. fusca by screening E. coli colonies containing plasmid
and phage libraries with T. fusca DNA inserts using a CMC
overlay assay22–24 and we cloned a xylanase gene using a xylan
overlay25. Since the four endocellulase genes that we cloned
encoded the four endoglucanases we had purified, it seemed
probable that we had identified all of the endocellulases that
are produced by T. fusca when it grows on cellulose. It is puz-
Fig. 1. Microscopic image of T. fusca micelia at a magnification of 60X.
zling that a second family 5 cellulase gene, which we did not
find, is present in T. fusca and recently it was shown to be
expressed in E. coli and have good activity on CMC26.
We succeeded in cloning the genes that encoded the two
teins including hair and feathers. We were able to isolate a T. fusca exocellulases, but we had to screen plasmid libraries
stable strain, ER1, which lacked the protease that we have used with labeled oligonucleotides designed to code for the N-
for most of our research6. Using standard purification methods, terminal sequence of each protein, which we had determined,
we were able to purify and characterize six different cellulases, as the enzymes had too low an activity on CMC to detect
a low molecular weight cellulose binding protein and a colonies containing their genes by a CMC overlay9,10. All of
xylanase from the culture supernatant of T. fusca ER1 grown the cloned genes were sequenced and the sequences showed
on cellulose7–10(Table I–II). In addition we also cloned puri- that every gene encodes a family 2 cellulose binding domain
fied and characterized an intracellular b-glucosidase11 and an (CBD), which is at the N-terminus of Cel5A, Cel6B and
extra cellular xyloglucanase12. A major contributor to all the Cel48A and at the C-terminus of Cel6A, Cel9A, Cel9B and
research discussed in this article is my long time coworker, Xyn11A (Fig. 2)27–29.
Diana Irwin. The T. fusca cellulase catalytic domains belong to four dif-
An important influence on our studies of the properties ferent families: 5, 6, 9 and 48. In the two families that con-
of T. fusca cellulases is a long standing collaboration with tained two cellulases, 6 and 9, the enzymes in each family are
Dr. Larry Walker in the Department of Biological and Envi- very different, as one family 6 cellulase is an endocellulase
ronmental Engineering at Cornell. He has carried out exten- (Cel6A) while the other is an exocellulase (Cel6B) and the cat-

 David B. Wilson was born in 1940 in Cambridge, MA, USA. He received his B.A. from
Harvard in 1961 and his Ph.D. from Stanford in 1966. He was a postdoctoral fellow in the
Biophysics Department at Johns Hopkins Medical School from 1966 to 1967. He joined Cornell
University as an Assistant Professor of Biochemistry in 1967, was promoted to Associate
Professor in 1973 and to full Professor in 1984. His current research continues to focus on
the mechanisms of plant cell wall degradation by bacterial enzymes. He was elected to the
American Academy of Microbiology in 2003. 

© 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. 73
THE CHEMICAL RECORD

Table I. Cellulases of Thermobifida fusca, activity of whole and truncated enzymes.

Activities (mmol CB/0 min-mmol)

Enzyme MW (kD) CMC SW FP BMCC Refs.


7
Cel 5A endo 46.3 2840 90 0.83 15
Cel 5Acd 34.5 2477 85.3 0.57* —
7,46
Cel 6A endo 43.0 355 631 0.85 6.5
Cel 6Acd 30.4 330 560 0.50* 3.2
9
Cel 6B exo-NR 59.6 0.3* 1.5 0.13* 2.0
Cel 6Bcd 45.7 0.5 1.1 0.05* 1.0
41
Cel 9A processive endo 90.4 475 202 1.03 19.1
Cel 9Acd + CBD3 68.0 488 54 0.24* 6.1
Cel 9Acd 51.4 108 6.3* 0.13* 0.15*
Cel 9Acd + CBD2 74.0 121 23.2 0.27* 0.29*
Cel 9A (D Fnll) 79.7 234 45.6 0.65 16.3
7
Cel 9B endo 101.2 5410 363 0.18* 0.42*
10
Cel 48A exo-R 104.0 0.3* 0.4* 0.07* 0.19*
Cel 48A cd 71.9 0.2* 0.2* 0.05* 0.09*
*target % digestion could not be achieved, assayed using 1.5 nmol/ml enzyme.
exo-NR = exocellulase, nonreducing end directed, exo-R = exocellulase, reducing end directed
endo = endocellulase

Table II. Other characterized carbohydrate active enzymes of T. fusca.

Enzyme MW (kD) Substrate Product Activity Km Ref.

mmol red. sugar/ min-mmol mg/ml


Xyn10B 42.3 Xylan—Birchwood mixed xylobiose 3,724 1.4 unpub
and higher MW
28
Xyn11A 31.9 xylan xylobiose and 15,600 1.1
higher MW
Xyn11Acd 19.9 xylan 14,200 2.3

mmol glu/min-mmol mM
11
Bgl1C 53.4 cellobiose pNp-glu glucose 1,497 0.35
719 0.24

mmol XGOs/min-mmol mg/ml


12
Xeg74 94.7 tamarind XG XGO’s mixed 578 3.2
Xeg74cd 79.5 875 3.9
XGO = xyloglucan oligomers
red. sugar = reducing sugar
pNp-glu = 4-nitrophenyl b-D-glucopyranoside

alytic domains have only 31% sequence identity. For family 9, all six T. fusca cellulases that are used to degrade cellulose are
one enzyme is an endocellulase (Cel9B), the other is a novel different from each other and appear to have been obtained
type of cellulase, a processive endoglucanase (Cel9A), and the from different organisms, rather than having been produced in
two catalytic domains have only 27% sequence identity. Thus, T. fusca by gene duplication.

74 © 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.
Microbial Cellulases

Fig. 2. Cartoon showing the arrangement of the domains of T. fusca glycohydrolases listed in Table I and II.

It is interesting that an unrelated mesophilic bacterium, cellulase genes from a common relative, as the location of the
Cellulomonas fimi, appears to utilize the same set of cellulases. family 2 CBD differs for five of the six enzymes (Table III). In
Its six cellulases have similar activities to those of T. fusca, and contrast, Streptomyces coelicolor, which grows weakly on cellu-
their catalytic and CBD domains are in the same families30. lose and lacks one of the six T. fusca cellulase genes, has the
However, the two organisms probably did not obtain their family 2 CBD encoded in the same location as in the corre-

© 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. 75
THE CHEMICAL RECORD

Table III. Cellulase families found in similar bacterial strains.

Thermobifida fusca Cellulomonas fimi Streptomyces coelicolor

CBD CBD % identity of CBD % identity of


Enzyme Type Location Type Location cd to T. fusca Location cd to T. fusca

Cel 5A Endo N-term Endo C-term 21 N-term 50


Cel 6A Endo C-term Endo N-term 42 C-term 49
Cel 6B Exo N-term Exo C-term 55 N-term 65
Cel 9A Processive C-term Processive C-term 80 — —
Cel 9B Endo C-term Endo N-term 59 C-term 60
Cel 48A Exo N-term Exo C-term 33 N-term 57

sponding five T. fusca genes (Table III). Thus, these related of Cel7A to T. fusca crude cellulase doubles its specific activ-
organisms appear to have obtained their cellulase genes from ity on crystalline cellulose8.
a common ancestor. At this time, it is not known why aerobic bacteria and
Studies of the activity of modified T. fusca cellulases, aerobic fungi use completely different enzymes to attack the
lacking the family 2 CBD, showed that this domain is not reducing end of cellulose. One possibility is that aerobic cel-
required for activity on the soluble substrate carboxymethyl lulolytic bacteria attack different types of cell walls than aerobic
cellulose (CMC) and it has only a small role in activity on fungi and that the family 48 enzymes are more active on the
amorphous cellulose, except for Cel9A. However, it is impor- cell walls that bacteria attack than the family 7 enzymes. We
tant for activity on crystalline substrates, such as filter paper or have started to test this idea by studying the digestion of
bacterial microcrystalline cellulose (BMCC) (Table I). Fur- tomato cell walls by T. fusca hydrolases. Preliminary studies
thermore, studies of the effect of the CBD on synergism have shown that a mixture of cellulases plus a xyloglucanase
showed that it is much more important for exocellulases than have low activity on plant cell walls, but that T. fusca crude
for endocellulases (Table IV). This work confirmed the impor- cellulase, which contains these enzymes along with other pro-
tant role that CBDs play in crystalline cellulose degradation teins, can degrade the cell walls12. Once we have identified the
and the presence of a binding domain appears to be a general minimal set of enzymes needed to degrade the cellulose in
adaptation for enzymes that degrade insoluble substrates, as plant cell walls, we can test the effect of replacing T. fusca
substrate binding domains have now been found on most Cel48A with T. reesei Cel7A in the hydrolysis of different types
enzymes that catalyze the hydrolysis of insoluble substrates31. of plant cell walls.
Another important finding from our cellulase research is Detailed studies of synergism by T. fusca cellulases, and by
the existence of two classes of exocellulases. One, with T. fusca cellulases plus T. reesei Cel6A and Cel7B have provided
members in family 7 and family 48, attacks the reducing ends strong evidence that synergism does not require specific inter-
of cellulose molecules and the other, with members so far only actions between the individual cellulases. It appears that two
in family 6, attacks the non-reducing ends32. Furthermore, we cellulases give synergism if and only if they attack insoluble
showed that two exocellulases only give synergism when they cellulose at different sites and if they create new sites for each
are from different classes. Our research also confirmed the other as a result of their activity. It is interesting that pretreat-
existence of bacterial exocellulases, which was first shown for ment of crystalline cellulose with an endocellulase makes it a
C. fimi, Cel6B33. The T. fusca exocellulase that attacks the non- better substrate for exocellulases36,37, but pretreatment with an
reducing end of cellulose chains, Cel6B, is very similar in its exocellulase does not increase the activity of endocellulases on
activity to the fungal exocellulases in this class. The main dif- the pretreated cellulose. We have shown that in synergistic mix-
ference is that the T. fusca enzyme is more thermostable and tures of endocellulases and exocellulases, the activity of the
has a broad pH optimum centered at pH 7, while the fungal endocellulase is increased8 so that the stimulation of the endo-
enzymes have narrow pH optima centered at pH 434. In con- cellulase must result from transient changes in the cellulose
trast, family 48 exocellulases have a very different structure structure, such as displacement of a segment of a cellulose
from the comparable fungal enzyme, T. reesei Cel7A35. Fur- strand, which returns to the original inaccessible state if the
thermore, Cel48A has very different activity, being much less endocellulase does not bind to it quickly. A careful study of
active than Cel7A, both by itself and in synergistic mixtures. the reason for the non-linearity of the rate of cellulose degra-
Cel7A is responsible for the higher activity of T. reesei crude dation by T. fusca cellulases with both time and amount of
cellulase, as compared to T. fusca crude cellulase, as addition enzyme38 showed that it was the heterogeneity of the substrate

76 © 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.
Microbial Cellulases

Table IV. Synergistic activity of binary mixtures of T. fusca enzymes on filter paper.

Molar Ratio Filter PaperActivity


Enzymes of Enzymes (mmol CB/min-mmol) References

Endo + Exo
8
Cel5A + Cel6B 1:4 2.85
Cel5Acd + Cel6B 1:4 2.15 ≤
Cel5A + Cel6Bcd 1:4 0.62* ≤
Cel5Acd + Cel6Bcd 1:4 0.63* ≤
7
Cel6A + Cel6B 2:1 3.8
Cel6Acd + Cel6B 2:1 2.0 ≤
9
Cel5A + Cel48A 1:4 0.76*
Cel6A + Cel48A 1:4 0.75* ≤

Exo + Exo
9
Cel6B + Cel48A 1:1 0.52*

Processive Endo + Endo


7
Cel9A + Cel5A 2:1 2.1
Cel9A + Cel6A 3:1 2.2 ≤

Processive Endo + Exo


9
Cel9A + Cel48A 1:4 1.8
7
Cel9A + Cel6B 1:1 2.0

Multiple Enzyme Mixtures


9
Cel9A + Cel5A + Cel48A 1:1:8 3.3
Cel6B + Cel9A + Cel5A + Cel48A 4:1:1:4 7.5 ≤
Cel9B + Cel6A + Cel6B + Cel9A + Cel5A 1:1:8:1:1 10.2 ≤
Cel9B + Cel6A + Cel6B + Cel9A + Cel5A + Cel48A 1:1:4:1:1:4 11.6 ≤
*5% digestion could not be achieved by these mixtures.

that caused the non-linearity, rather than product inhibition endocellulases produce between 30 and 40% insoluble prod-
or cellulase instability. ucts from filter paper8. The structure of Cel9Acd, determined
In a collaboration with Dr. Andrew Karplus at Cornell, by Dr. Sakon in Dr. Karplus’ laboratory, contains a family 9
the 3-dimensional structure of the T. fusca Cel6A catalytic catalytic domain rigidly attached to a family 3c CBD, with the
domain was determined by X-ray crystallography39. The struc- active site cleft in the catalytic domain aligned with the poten-
ture was ultimately refined to 1.18Å and it was the highest res- tial cellulose binding site on the CBD (Fig. 4)41. The structure
olution structure of a 30,000 MW protein when published of Cel9Acd was also determined with several oligosaccharides
(Fig. 3). The structure showed that Cel6A, an endocellulase, bound in the active site. Comparison of liganded structures
has an open active site cleft as had been predicted by Rouvi- with the free structure identified the catalytic water in the free
nen et al.40, who had published the first cellulase structure, that structure and showed that several residues moved upon sub-
of Trichoderma reesei Cel6A, an exocellulase, with its active site strate binding41. The enzyme appears to make an initial endo-
in a tunnel. At this time, these structural differences have been cellulolytic cleavage of a cellulose molecule, and then the
found for all true endocellulases and exocellulases, although in fragment bound to the CBD slides into the active site and
some exocellulases only part of the active site cleft is enclosed35. cellotetraose units are cleaved processively from the non-
A major finding from our research is the identification of reducing end of the bound chain. This mechanism was con-
a new type of cellulase, Cel9A, which is a processive endoglu- firmed by constructing two other forms of Cel9A using genetic
canase. This enzyme has relatively high activity on CMC, like engineering. One lacked the family 3 CBD, while the other
an endocellulase but it also produces 87% soluble products contained only the catalytic domain. The activities of the
from filter paper and only 13% insoluble products, while true constructs (Table I) show that removal of the family 3 CBD

© 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. 77
THE CHEMICAL RECORD

Fig. 3. Cel 6A structure made using PDB file 1tml with cellotetraose (red)
modeled into the open cleft active site. Asp 117 (black) is the catalytic acid.
Other important catalytic residues are Asp 79 (cyan), Arg 78 (pink), Tyr 73
(purple), Asp 156 (gray), and Asp 265 (blue)48,49.

Fig. 4. Cel9A structure using PDB file 4tf4 showing six glucose residues in
the active cleft (red) and the beta strand 2 (Thr475 to Gln 486) of the CBD3c
in solid 3D (gold). The region deleted for the block mutant, Thr245- Arg252
converts Cel9A into a true endocellulase, completely abolish- is colored black. The calcium atoms are shown in black; Glu424, the catalytic
ing its processive activity as predicted by our mechanism42. acid, is blue. Asp55 and Asp58, the putative catalytic bases, are shown in
This type of enzyme has been found in both anaerobic and purple. An extended cellulose chain could interact well with the conserved
residues of beta strand 2 and the active cleft of the catalytic domain. The
aerobic bacteria, but not in eucaryotic organisms43,44. Cel9A product is released from the Glc(-1) to Glc(-4) sites41.
can give synergism with endocellulases and both classes of exo-
cellulases, providing further evidence that Cel9A is a unique
type of cellulase (Table IV).
We have carried out extensive site-directed mutagenesis ing Ser85 to Ala has no effect on activity, yet in their model
studies on Cel6A, both to determine its catalytic mechanism the equivalent residue is hydrogen bonded to the catalytic
and to try to determine how it hydrolyzes crystalline cellulose. water molecule. This result shows that either the two enzymes
This enzyme is ideal for these studies as we have a high- use somewhat different mechanisms or their model, which is
resolution structure of the catalytic domain as well as a struc- based on detailed structural information, is wrong, which
ture with cellobiose bound in subsites Glc(-2) and Glc(-1). seems unlikely.
Furthermore, Cel6Acd is expressed in E. coli at a high level Some of the Cel6A mutations reduce activity on CMC
(170 mg/l) so that it is relatively simple to produce the mutant and amorphous cellulose to a much greater extent than activ-
enzymes. Finally it has been possible to determine the Kd for ity on crystalline cellulose. This shows that the rate limiting
both methylumbelliferyl glycoside and oligosaccharide binding step for crystalline cellulose hydrolysis differs from that of the
to Cel6A by fluorescence quenching45. At this time, we have other substrates. This is not surprising since Cel6A has a spe-
constructed fifty-eight mutants in 33 of the 306 total residues cific activity on crystalline cellulose that is 1/500 that on the
in this protein46–48. In addition to determining the activities of other substrates. I believe that the rate limiting step on crys-
the mutant enzymes on different types of cellulose and some talline cellulose is probably the binding of a cellulose chain into
oligosaccharides, we determined their binding affinities to the active site and that once it is bound it is rapidly cleaved.
several oligosaccharides and methylumbelliferyl glycosides. A key question is which residues function in the rate limiting
The results from all these studies combined with molecular step? At this time, we only have succeeded in finding one
dynamics modeling of Cel6A bound to cellotetraose allowed mutation that reduces activity on crystalline substrates to a
us to propose a detailed mechanism for cellulose hydrolysis by greater extent than it reduces activity on other substrates,
Cel6A (Fig. 3)49. Surprisingly, our model differs slightly from which is the result expected for a residue that only participates
the model proposed for T. reesei Cel6A50, as we find that mutat- in the rate limiting step. Even with this residue, only one of

78 © 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.
Microbial Cellulases

Table V. Cel6A mutants resulting in higher CMC activity. Further evidence for loop movement comes from mea-
surements of the thermodynamics of MU(Glc)2 binding to
Cel6A mutant % CMC Activity46 Cel6Acd and two family 6 cellulases where the loop is less
mobile. For Cel6A, DS = –46.8 (J/mol K)(41), while for T.
wild type 100
fusca Cel6B and T. reesei Cel6A the values are 53.4 and 63,
Arg 237 Ala 176
Glu 263 Asp 236 respectively51. This dramatic difference can be explained if sub-
Glu 263 Gln 162 strate binding restricted the mobility of the residues in the
Glu 263 Gly 110 Cel6A loop, thus decreasing the entropy while there is only a
Lys 259 His 373 small change in the mobility of the loop in the exocellulases.
Arg 237 Ala, Glu 263 Asp 290 Dr. Anna Larsson in Alwyn Jones’ laboratory has deter-
Arg 237 Ala, Glu 263 Gln 363 mined several new crystal structures for T. fusca Cel6A WT
His 159 Ser 168 and the mutant enzymes (personal communication). The wild-
Lys 259 His 485 type structure differs from our original structure in that most
of the loop (residues 81–87) is not visible as it is too mobile,
but the rest of the molecule has a structure that is the same as
three amino acid substitutions showed this effect. One very the original one. A second structure is of a mutant enzyme
interesting mutation, Trp16 to Ile, lowered activity on amor- Tyr73Ser that has extremely low activity. The structure of this
phous cellulose more than activity on CMC or filter paper. mutant enzyme was also determined with a cellotetraose ana-
This differential effect was increased in the catalytic domain. logue bound in the active site. This structure also lacks residues
The structure of this mutant cd was determined by X-ray crys- 81–87, possibly because the mutation prevents the conforma-
tallography and the mutation created minor shifts in a number tional change, explaining the loss of activity. A structure of the
of regions (A. Larsson, personal communication). Asp117Ala mutant enzyme crystallized in the presence of cel-
We identified five residues where mutations produce lotriose shows a glucose residue bound in the Glc(–3) subsite
enzymes with significantly higher activity on the soluble which is the first evidence for this subsite in Cel6A.
substrate carboxymethyl cellulose (Table V)47. Every mutant A similar conformational change has been shown to occur
enzyme, which has higher activity on CMC, has weaker in two family 5 endoglucanases, Clostridium thermocellum
binding to oligosaccharides. Unfortunately, none of the CelC52 and Humicola insolens endoglucanase 553. In each
mutant enzymes have increased activity on insoluble enzyme, the loop in the unliganded structure is less ordered
substrates. than in the liganded structure and the loop moves to partially
There are a number of experiments that show that the cover the substrate in the active site cleft. Family 5 and family
loop in Cel6Acd from residue 73 to 87 undergoes a major con- 6 enzymes are similar in structure and both loops contain two
formational shift during or after substrate binding and that this adjacent Gly residues at one end.
shift is essential for catalysis. Furthermore, the position of the We also have made site-directed mutants in the exocellu-
loop in the X-ray structure is not that of the active conforma- lase Cel6B, using a model structure based on the structures
tion. The evidence that the structure is not the active structure of the related enzymes, T. fusca Cel6A and T. reesei Cel6A.
of Cel6A is the fact that mutation of Asp79 to Asn causes a Twenty-three mutant genes in eleven different residues were
30–50 fold reduction in activity on CMC or amorphous cel- constructed and the expressed proteins were purified and char-
lulose48, even though in the X-ray structure, Asp79 is 11.3Å acterized51. Two pairs of mutations were constructed to create
from the site of bond cleavage. In T. reesei Cel6A, an exocel- new disulfide bonds that joined the two loops, which form the
lulase, the corresponding residue is only 7Å from the site of active site tunnel (N233C/0506C and 230C/0512C). Both
cleavage40. mutant enzymes contained the new bond and both retained
One experiment that shows that the conformational activity on all substrates tested, showing that opening of the
change is essential for activity is mutation of either of two adja- loops is not required for activity as had been suggested54.
cent Gly residues (86, 87) at one end of the loop to Ala, as Unfortunately, neither mutant enzyme showed increased ther-
both mutations significantly decrease Cel6A activity (16 and mostability, as has been seen when a new disulfide bond was
4.5% residual activity, respectively)46. Since these residues are created in some proteins. Four Gly residues were mutated to
not buried and are not in the active site, the main effect of try to increase thermostability51, but none of them did. Sur-
either mutation is to reduce the ease of loop movement by prisingly, two of these mutations increased activity on FP and
increasing the size of the side chain. Removal of the disulfide BMCC and a double mutant containing both mutations had
bond containing another loop residue, by mutating both even higher activity on FP (twice wild type (WT)). Unfortu-
Cys80 and Cys125 to Ser also reduced activity (12% residual nately, this increase in activity did not show up in synergistic
activity), possibly by changing the final position of the loop46. mixtures with an endoglucanase. Several mutants had signifi-

© 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. 79
THE CHEMICAL RECORD

cantly reduced inhibition by cellobiose, but they also had tion in the CelR gene that lowered its affinity for the 14 base
slightly lower activity than the WT enzyme. sequence providing conclusive evidence that CelR functions in
Site-directed mutagenesis has been used to determine the the regulation of cellulase synthesis63. There are two findings
role of eight key residues in Cel9A and two deletion mutants that suggest that CelR is not just a repressor. One is the fact
were constructed55. One removed a FnIII domain (Fig. 2), that the two exocellulase genes contain multiple copies of the
while the other removed seven residues that block one end of CelR binding sequence, which are located at different
the active site cleft (Fig. 4). The properties of the point mutants upstream sites from the single endocellulase sites and the exo-
confirmed that Glu424 functions as the catalytic acid and that cellulases are made in equal amounts while each of the endo-
both Asp55 and Asp58, which bind the catalytic water, are cellulases is present at 1/3 to 1/6 the level of an exocellulase.
required for the catalytic base function. These residues are con- There are no strong similarities in the potential cellulase pro-
served in all members of family 9 and this is the only hydro- moter sequences, so it is possible that CelR regulates the level
lase family in which two acidic residues bind the catalytic of synthesis of the cellulases. The other very surprising finding
water. When Tyr206 was replaced by Phe, Cel9A retained is that the level of CelR is much lower in T. fusca grown on
some activity and binding, but when it was replaced by Ser, glucose or xylan than in T. fusca grown on cellobiose or cellu-
activity and binding were lost. Mutation of Tyr318 greatly lose. We are still working to understand exactly how CelR
reduced binding, but only partially reduced activity. However, functions and if it is participating in both controls.
both of these mutant enzymes were non-processive, showing The T. fusca genome was sequenced in 2000 by the
that high affinity binding in the Glc(-3) to Glc(-1) subsites is U.S. Department of Energy Joint Genome Institute
essential for processivity. Removal of the FnIII domain (http://www.jgi.doe.gov/JGI_microbial/html/index.html). It
decreased activity and processivity to 65% of wild type and is 3.7 MB in size and in addition to the six cellulase genes we
decreased binding to BMCC slightly. When the two FnIII had identified, it contained a second family 5 cellulase gene
domains were removed from Clostridium thermocellum CbhA, and a family 74 gene, but neither of these genes contain a
a family 9 exocellulase, activity was reduced to 50%56. It is not perfect upstream CelR binding site. We have expressed and
clear how this domain affects activity but its primary role seems characterized the family 74 enzyme and it is a xyloglucanase
to be in the digestion of crystalline cellulose. Evidence for (Xeg74) with low activity on CMC12. It is produced by cells
FnIII domain activity disrupting crystalline cellulose was also grown on cellulose, xylan or corn fiber, but not by cells grown
presented by Nidetzky et al.57 Deletion of the block at the end on glucose or cellobiose. Xeg74 contains a family 2 CBD and
of the T. fusca Cel9A active site cleft did not change activity it appears to function to remove xyloglucan from cellulose in
or products but did increase binding to BMCC. This result plant cell walls, allowing the cellulases better access to the cel-
shows that production of cellotetraose during processive lulose (Fig. 5)12. T. fusca can not grow on xyloglucan as a
hydrolysis does not result from the peptide block. carbon source, even though it completely degrades it to
T. fusca regulates cellulase synthesis in two ways, induc- oligosaccharides. It seems likely that a xylanase cloned from
tion by cellobiose and carbon source repression58–60. Induction T. alba64 which has a family 2 CBD, has a similar function,
increases the cellulase level about 12 fold and derepression removing xylan to allow the cellulases better access to the cel-
increases the level about nine fold. Growth on cellulose gives lulose in plant cell walls, as its synthesis is induced by cellobiose
the highest cellulase level as it simultaneously induces and and not by xylan. T. fusca contains a number of other xylanase
derepresses, since the products of cellulose degradation are genes that are induced by xylan and these probably function
consumed by T. fusca as rapidly as they are produced. Both to allow T. fusca to grow on xylan65.
mechanisms act at the level of transcription for Cel5A, the only While much of my cellulase research has focused on T.
enzyme that was tested59. All of the six T. fusca cellulase genes fusca cellulases, I have collaborated with Dr. James Russell in
contain the same upstream fourteen base inverted repeat the Department of Microbiology at Cornell to study cellulose
sequence TGGGAGCGCTCCCA. In the four endocellulase digestion by rumen bacteria. We started using genetic engi-
genes, there is a single copy of this sequence, which starts neering to try to construct a cellulolytic rumen bacterium
between 36 and 63 bases upstream of the start codon, while that could degrade cellulose at pH values below 5. Prevotella
in the two exocellulase genes there are two copies that are from ruminicola was chosen as the starting organism as it grows
200 to 350 bases from the start codon60. This sequence is the readily on cellulodextrins at low pH. We cloned, expressed and
binding site for a regulatory protein, CelR, that has been characterized a family 5 endoglucanase gene from P. rumini-
cloned, expressed, purified and characterized61,62. This protein cola66, which did not encode a CBD and had low activity on
is a member of the lactose repressor family and it binds cel- insoluble cellulose. Further work showed that the enzyme
lobiose with a mM Kd62. Furthermore, cellobiose lowered the expressed in E. coli was only the C-terminal domain of the
affinity of CelR to DNA containing the 14 base sequence. A P. ruminicola protein and that P. ruminicola produced two
mutant that was constitutive for cellulase synthesis had a muta- endoglucanases that only differed in their N-terminal

80 © 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.
Microbial Cellulases

TFSF-GBX crystalline cellulose, allowing it to be hydrolyzed by


TFSF-GBG endocellulases70.
Cel Mix - GBX
300 Cel Mix + XEG74 - GBX
The T. fusca research described in this paper has been
mg reducing sugar produced

250 supported throughout by the U.S. Department of Energy,


Basic Energy Sciences Program and additional support has
200
come from the D.O.E. National Renewable Energy
150 Laboratory and the USDA competitive Grants Program.

100

50
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82 © 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.