Cloning, sequencing and homologies of the cbh-1 (exoglucanase) gene of Humicola grisea var. thermoidea

Abstract

Studies on the enzymes of the cellulase complex of the thermophilic fungus Humicola grisea var. thermoidea are described. A genomic library was constructed in the phage vector EMBL 4, and from this library two clones were isolated using as a probe the cloned cbh-1 (exoglucanase, EC 3.2.1.91) gene of Phanerochaete chrysosporium, a cellulolytic basidiomycete fungus. These clones were analysed by restriction mapping and Southern blotting, and one of them (lambda 3) was sub-cloned into the M13 phage vectors mp18 and mp19. The gene sequence was determined by the dideoxy chain-termination method. Sequence comparison with the equivalent genes from P. chrysosporium and Trichoderma reesei was made: in terms of primary sequence there is about 60% homology between the three species. Secondary structure prediction of the H. grisea sequence was also computed.

Full text

Journal
of
General Microbiology
(1990),
136,
2569-2576.
Printed in Great Britain
2569
Cloning, sequencing
and
homologies of the
cbh-1
(exoglucanase) gene of
-
Humicola
grisea
var.
thermoidka
MARISTELLA
DE
0.
AZEVEDo,'t MARIA
S.
S.
FELIPE,2t SPARTACO ASTOLFI-FILH02t
and
ALAN
RADFORD~"
'Department
of
Genetics, The Uniuersity
of
Leeds, Leeds
LS2
9JT,
UK
Department
of
Biochemistry, University
of
Manchester Institute
of
Science and Technology,
PO
Box
88,
Manchester
Mi50
lQD,
UK
(Receiued
5
March 1990; revised
30
July 1990; accepted
20
August 1990)
Studies
on
the
enzymes
of
the
cellulase
complex
of
the thermophilic fungus
Humicola
grisea
var.
thermoidea
are
described.
A
genomic library
was
constructed in the phage vector
EMBL
4,
and from this library
two
clones were
isolated using
as
a
probe
the cloned
cbh-1
(exoglucanase,
EC
3.2.1- 91) gene of
Phrochaete chrysosprium,
a
celldolytic
basidiomycete
fungus.
These
clones were
analysed by
restriction mapping and Southern blotting, and
one
of
them
(iU)
was
sub-cloned into the M13 phage
vectors
mpl8 and mp19.
The
gene sequence
was
determined
by
the dideoxy chain-termination method. Sequence comparison
with
the
equivalent
genes
from
P.
chrysosporium
and
T&hu&rma
reesei
was
made: in
terms
of
primary sequence there
is
about
60%
homology
between
the three
species.
Seconaary structure prediction
of
the
H.
grisea
sequence
was
also
computed.
Introduction
Humicola grisea
var.
thermoidea
is
a thermophilic
imperfect filamentous fungus. The isolate used in this
work is an efficient cellulolytic organism, producing high
levels of the enzymes of the cellulase complex, namely
endoglucanases (EC 3.2.1.4), exoglucanases (EC
3.2.1
.91)
and p-glucosidase (EC 3.2.1 ,21) (Araujo
et
al.,
1983;
R.
Monti, personal communication). The
enzymes of the cellulase complex have commercial
potential as well as considerable scientific interest.
Cellulolytic enzymes have been studied in other species
of fungi, such as
Trichoderma reesei
(Shoemaker
et al.,
1983
;
Teeri
et al.,
1983),
Phanerochaete chrysosporium
(Sims
et al.,
1988) and
Neurospora
crassa
(Eberhardt,
1961; Eberhardt
&
Beck, 1973; Yazdi
et
al.,
1990a,
b).
Some
of
the cellulase complex genes of
T.
reesei
and
P.
chrysosporium
have been cloned and sequenced.
In general, the endoglucanases and exoglucanases are
exported enzymes because of the insolubility of their
substrate, cellulose, but the /3-glucosidases, which further
t
Present address: Department of Cellular Biology, The University
of
Brasilia, Brasilia,
DF,
Brazil.
The nucleotide sequence data reported in this paper have been
submitted to EMBL and have been assigned the accession number
X17258.
degrade the cellobiose resulting from the action of the
glucanases, are predominantly intracellular (Gong
&
Tsao, 1980). Endoglucanases and exoglucanases have a
characteristic domain structure, with the greater part of
the sequence specifying
a
catalytic domain, separated
from a cellulose-binding domain by a 'hinge' rich in
serine or proline and threonine residues (Knowles
et al.,
1987).
Here we describe the analysis, by gene cloning and
sequencing, of an enzyme of the cellulase complex
of
H.
grisea
var.
thermoidea.
Methods
Fungal strain.
The strain of
H.
grisea
var.
thermoidea
used was
a
wild
isolate obtained in Brazil (Araujo
et al.,
1983).
It was routinely maintained on slants of 4% (w/v) oat flour,
1.5%
(w/v) agar, on which
good
conidiation was achieved after 5
d
at 42
"C.
For the production of mycelium, a rich liquid medium was used, based
on the minimal medium of Pontecorvo
et al.
(1953), supplemented with
0.2% peptone, 0.15% hydrolysed casein, yeast extract, yeast nucleic
acid and vitamin solution, adjusted to pH 6.8 (Azevedo
&
Costa, 1973).
Phage vectors and bacterial strains.
The phage vector in which the
library was constructed was EMBL4 (Frischauf
et al.,
1983). Sub-
cloning for DNA sequencing was into the M13 phage vectors mp18 and
mp19 (Messing, 1983
;
Yanisch-Perron
er
al.,
1985).
Escherichia coli
host
strains used were TG1, L2392 (Maniatis
et al.,
1982) and P2392, a
derivative of L2392 (Perbal, 1989).
0001-6122
0
1990
SGM

2570
M.
de
0.
Azevedo and others
DNA
extraction. Mycelium was grown overnight at 42 "C in the rich
liquid medium from a large inoculum of conidia (about lo5 conidia
ml-l)
from a 7-14-d-old culture. The mycelium was harvested by
filtration, and stored at
-
80
"C. A 2 g aliquot of mycelium was ground
under liquid nitrogen, resuspended in 15
ml
TTE buffer (10 mwTris
pH 8-5, 10 mM-EDTA, 4 m-spermidine, 10 m~-2-mercaptoethanol,
0.5 M-Sucrose, 36 mM-KC1, 0.25% Triton X-100), and centrifuged at
2500
g
for 10 min. The pellet was carefully resuspended in lysis buffer
(40
mM-Tris pH
8-0,lO
m~-EDTA, 0.2 M-NaCl, 1.5%, w/v, SDS), and
extracted with double-distilled phenol liquified with buffer (Maniatis et
af.,
1982). The aqueous phase was extracted a second time with phenol,
and then with chloroform. Then 0.1 vol. 3 wsodium acetate
(PH
5-5)
was added, and the mixture was ethanol-precipitated at
-
20 "C. The
precipitated DNA was spooled out with a glass
rod,
washed with 70%
(v/v) ethanol, dried at room temperature, and redissolved in TE buffer
(10 mwTris, 10 m~-EDTA;
pH
8.0)
to a final concentration of about
0.5
pg/ p1-1.
Construction
and
screening
of
the genomic library. The H. grisea
genomic library was constructed by completely digesting the
L
phage
vector EMBLA (Promega Biotech) with BamHI and
Sun,
and making a
partial digest of
H.
grisea genomic DNA with Sau3A to an average
fragment length of 15 kb. Fragments in the size range 9-18 kb were
ligated into the cut vector, the ligation mixture packaged with a
L
packaging kit (Gigapack) according
to
the manufacturer's instructions,
and the packaged mixture plated onto
E.
coli
L2392 (Maniatis
et
al.,
1982). The gene library was amplified in
E.
coli P2392.
The library was screened by plaque hybridization, using
E.
coli
P23W as host. The probe employed was a 1.7 kb PstI genomic fragment
containing a clone of the Phanerochaete chrysosporium cbh-1 gene (Sims
et
al.,
1988). The probe was labelled with [P~~PMATP, using a random
primer kit (Boehringer Mannheim) according
to
the manufacturer's
instructions.
After plating an aliquot of the library on P2392 according to
Maniatis et
al.
(1982), and incubating overnight at 37 "C, the plates
were blotted onto nitrocellulose filters (Schleicher and Schuell),
denatured for 2min, and then neutralized. The filters were then
washed, baked, incubated with pre-hybridization solution, hybridized
with the probe and analysed by autoradiography. Plaque areas giving a
good positive signal were identified, replated at lower plaque density,
and individual positive plaques identified and isolated (Maniatis et
al.,
1982).
Positive clones were further investigated by restriction and Southern
blotting. DNA from these was prepared, and cut with EcoRI, BamHI
and
SalI,
singly and in double digests, and run
on
agarose gels, with
standard size markers (HindIII-cut
L
and HaeIII-cut 4x174). After
denaturing the DNA, the gels were blotted onto nitrocellulose
membranes (Schleicher and Schuell), transferring overnight in 10
x
SSC
(1
x
SSC is 0.15 M-NaC1, 0.015 M-sodium citrate, pH 7.0). The
membranes were baked, incubated in pre-hybridization solution,
incubated with probe, washed, and analysed by autoradiography.
Sub-cloning and sequencing. For sub-cloning into M13 vectors mp18
and mp19, vector DNA was digested with EcoRI/SalI. Either total
or
agarose-gel-purified clone DNA was then digested with EcoRI/SalI
and ligated into the cut vector DNA. Competent cells of
E.
coli
TGl
were prepared according to Chung et
al.
(1989) or Mandel
&
Higa
(1970), and transformed with the ligation mixture according
to
the
method of Chung et al. (1989). After transformation and plating on
medium containing IPTG and X-Gal, white plaques were isolated.
From these, replicative-form DNA was prepared, digested, run on an
agarose gel, and probed with the
P.
chrysosporium cbh-l clone to
identify those sub-clones containing inserts of interest. Sequencing was
done by using the dideoxy chain-termination method (Sanger et
al.,
1977) and T7 DNA polymerase, in kit form (Pharmacia).
The software
of
Staden (1982) was used to compile and translate the
gene sequence. Alignment of the H. grisea sequence with those of the
equivalent
T.
reesei and
P.
chrysosporium genes was carried out with
MANALIGN,
one of a suite of sequence analysis programs in the
OWL
database and associated suite
of
analytical software (Akrigg et
at.,
1988). Secondary structure was computed with
PREDICT,
another
program written for the
OWL
database, which integrates the methods of
both Chou
&
Fasman (1978) and Gamier et
al.
(1976).
Results
and
Discussion
A Sau3A partial digest of
H.
grisea genomic DNA of size
range 9-18 kb was ligated into BamHIISaZI-cut
EMBU
vector
DNA.
This ligation mixture was mixed with cells
of
strain P2392, and plated. A total of
3
x
lo6
plaques
were obtained from 03ml
of
the packaged mixture,
harvested and stored. For an organism with.a genome of
about
lo7
bp and inserts of
10-15
kb, coverage of the
genome in the library at a probability of 0.99 requires
15
000
clones.
To screen the
H.
grisea genomic library with the
P.
chrysosporium cbh-l probe, approximately
6
x
104
plaques on a total of six 9 cm diameter plates were
blotted in duplicate onto nitrocellulose filters, and
probed with [32P]dATP-labelled isolated insert. Thirty
positive plaques were detected. These were isolated,
replated at low plaque density, and re-screened. Two of
them, identified as 13 and A9, gave clear and uniform
positive hybridization in this re-screening, and were
selected for further study.
Restriction mapping of clones ;/3 and A9 showed them
to be different in their restriction patterns with
EcoRI,
13
map
cbh-1
'S
SF
E
SB
B
I
I1
I
I
1
I
I
II
I
I
I I
I
2.6
I
3.7
I
I
2.8
I
I
L
0.8
I
~
I
6.4
I
A9
map
S
EB
S
B
E
I
II
I
I
I
I
II
1
I
I
1
2.2
I
1
2.6
I
I
8.0
I
Fig.
1.
Restriction maps of the
H.
grisea clones A3 and 19. In each case
the bold line indicates the region to which the
P.
chrysosporium probe,
essentially the entire coding region
of
the cbh-1 gene, hybridized. The
restriction sites indicated are those spanning the fragments to which
the probe bound, and hence the closest sites for EcoRI
(E),
SalI
(S)
and
BamHI'CB) on either side of the gene. Distances are given in kb.

A
cellulase gene
of
Humicola grisea
2571
Exon
1
Intron
ATG
Exon
2
Sal
I
TAA
Fig.
2.
The sequencing strategy employed for
H.
&sea
cbh-1.
The figure indicates the total length sequenced, the positions
of
the intron
and (bold lines) two exons, and the location
of
the
Sufi
site in the second exon. The arrows below indicate the positions
of
sequencing
primers, and the direction and extent
of
sequence determined
from
each.
For
the initial sequences out from the
Safi
site,
M13
‘-40’
primer was used.
(=s
specific primers).
BamHI
and
San.
The restriction gel was blotted, and
probed with the isolated
P.
chrysosporium cbh-1
clone
labelled with [32P]dATP. With one exception, this gave a
single hybridizing band with each single restriction
digest. In the case of clone
A3
cut by
Sari,
two bands
hybridized, indicating a restriction site within the region
of
A3
to which the probe was hybridizing. As the
P.
chrysosporium
probe contained little other than the
coding region of the
cbh-1
gene, this indicated a probable
SalI site within an equivalent gene in
A3.
Fig.
1
illustrates
restriction maps of clones
A3
and
19,
based on the sizes of
the hybridizing restriction fragments.
It is clear from the data in Fig.
1
that clones
A3
and
A9
are different in their restriction patterns, but they show
approximately equal levels of similarity to the probe,
based on the efficiency of hybridization. As is shown
below,
A3
contains a gene that is clearly homologous to
the
cbh-1
genes of
T.
reesei
and
P.
chrysosporium.
A9
has
not yet been further characterized, but its high level of
hybridization to the probe suggests that it may well
contain another gene of the cellulase complex. In support
of this conclusion, the homology between the
cbh-1
(exo-
glucanase) and
eg-1
(endo-glucanase) genes of
T.
reesei
is
quite high in parts of their sequence, high enough to use
one as a probe for the other.
Based on the above restriction and hybridization data,
EcoRI/SalI double digest fragments of
A3
were sub-
cloned into mp18 and mp19, the ligation mixture
transformed into
E.
coli
strain TG1, and sub-clones
detected as white plaques on X-Gal plates. Inserts of
interest were identified by probing with the
P.
chryso-
sporium cbh-1
fragment.
As the restriction analysis had shown an internal
SalI
site,
A3
sub-clones which had a
SalI
site adjacent to the
primer site were chosen for sequencing. From this initial
sequencing, reading was extended further with custom
oligonucleotide primers until the coding region had been
traversed. Complementary sub-clones were then used to
read back on the second strand to, and through, the
starting
SalI
site. The entire sequencing strategy is
shown in Fig. 2.
Data from the first sequencing gels, reading out from
the internal
SalI
site, were translated in all six possible
reading frames, and the homology with the
T.
reesei
and
P.
chrysosporiurn cbh-1
gene sequences at the amino-acid
level was immediately apparent. This identified unequi-
vocally the correct reading frame, orientation and
approximate location
of
the sequenced region within the
gene. Oligonucleotides
(1
7-mers) corresponding to the
furthest unambiguous sequence from each reading were
synthesized, and used as primers to extend the reading on
the same sub-clones. This process was repeated until the
entire open reading frame and beyond had been
sequenced. Complementary 17-mer primers were used
for sequencing the second strand back to the internal
SaZI
site, using further appropriate sub-clones. Alto-
gether 2.046 kb of sequence was determined (Fig.
3).
Analysis of the DNA sequence of
A3,
and computer
translation of a large open reading frame (Azevedo
&
Radford, 1990), together with preliminary comparisons
with the
cbh-1
genes of
T.
reesei
and
P.
chrysosporium
revealed a coding region of
1575
nucleotides, containing
two exons interrupted by a single, small intron of
60
nucleotides. The predicted translation product of the
open reading frame would have an
M,
of 55654, but this
does not take into account the effect of probable
glycosylation of such an extracellular enzyme. The first
exon starts with a typical N-terminal signal sequence of
about 18 amino acid residues, with the basic residues
arginine and lysine near the N-terminus followed by
a
short region rich in hydrophobic residues. The intron is
of a length typical for filamentous ascomycete nuclear
gene introns. It is defined by the
5’
consensus GTA and
the
3’
consensus
CAG,
and contains, starting 21
nucleotides upstream from the
3’
end, the internal
consensus GCTAAC. This intron does not correspond in
position to those found in the equivalent gene
of
the other
two species. If it were to be translated, this intron would

2572
M.
de
0.
Azevedo and
others
gccgtgaccttgcgcgctttgggtg8cggtggcgagtcgtggacggtgcttgctggtcgccggccttccc
ggcgatccgcgtgatgagagggccaccaacggcgggatgatgctccatggggaacttccccatggagaag
agagagaaacttgcggagccgtgatctggggaaagatgctccgtgtctcgtctatataactcgagtctcc
ccgagccctcaacaccaccagctctgatctcaccatccccatcgacaatcacgcaaacacagcagttgtc
gggccattccttcagacacatcagtcaccctccttcaaaATGCGTACCGCCAAGTTCGCCACCCTCGCCG
ALVASAAAQQRCSLTTERHPSLSW
CCCTTGTGGCCTCGGCCGCCGCCCAGCAGGCGTGCAGTGCAGTCTCACCACCGAGAGGCACCCTTCCCTCTCTTG
MRTAKFATLA
KKCTAGGQCQTVQASITLDSNWR
GAACMGTGCACCGCCGGCGCCAGTGCCAGACCGTCCAGCTTCCATCACTCTCGACTCCAACTGGCGC
WTHQVSGSTNCYTGNKWDTSICT
TGGACTCACCAGGTGTCTGGCTCCACCAACTGCTACACGGGCAACAAGTGGATACTAGCATCTGCACTGA
DAKSCAHNCCVDGADYTSTYGITT
TGCCAAGTCGTGCGCTCAGAACTGCTGCGGTCGATGGTGCCGACTACACCAGCACCTATGGCATCACCAC
NGDSLSLKFVTKGQHSTNVGSRT
CAACGGTGATTCCCTGAGCCTCAAGTTCGTCACCAAGGGCCAGCACTCGACCAACGTCGGCTCGCGTACC
YLMDGEDKYQ
TACCTGATGGACGGCGAGGACAAGTATCAGA~tacgttctatcttcagccttctcgcgccttgaatcctg
TFELVGNEFTFDVDVSN
pctaacqtttacacttca~CCTTCGAGCTCCTCGGCAACGAGTTCACCTTCGATGTCGATGTCTCCAA
IGCYLNGALYFVSMDADGGLSRY
CATCGGCTGCGGTCTCAACGGCGCCCTGTACTTCGTCTCCAT~ACGCCGATGGTGTCTCAGCCGCTAT
PGNKAGAKYGTGYCDAQCPRDIK
CCTGGCAACAAGCTGGTGCCAAGTACGGTACCGGCTACCGGCTACTGCGATGCTCAGTGCCCCCGTGACATCAAGT
FINGEANIEGWTGSTNDPNAGAGR
TCATCAACGGCGAGGCCAACATTGAGGGCTGGACCGGCTCCACCAACGACCCCAACGCCGGCGC~GCCG
YGTCCSEMDIWEAQQHATAFIPH
CTATGGTACCTGCTGCTCTGAGATGGATATCTGGGAAGCCAACAACATGGCTACTGCCTTCACTCCTCAC
PCTIIGQSRCEGDSCGGTYSNER
CCTTGCACCATCATTGGCCAGAGCCGCTGCGAGGGCGACTCGTGCGGTGGCACCTACAGCAACGAGCGCT
YAGVCDPDGCDFNSYRQGNKTFYG
ACGCCGGCGTCTGCGACCCCGATGGCTGCGACTTCAACTC~TACCGCCAGGGCAACAAGACCTTCTACGG
KGMTVHTTKKITVVTQFLKDANG
CAAGGGCATGACCGTGCACACCACCAAGAAGATCACTGTC~TCACCCAGTTCCTCAAGGATGCCAACGGC
DLGEIKRFYVQDGKIIPNSESTI
GATCTCGGCGAGATCAAGCGCTTCTACGTCCAGGATGGCAAGATCATCCCCAACTCCGAGTCCACCATCC
PGVEGNSITQDWCDRQKVAFGDID
CCGGCGTCGAGGGCAATTCCATCACCCAGGACTGGTGCGACCGCCAGAAGGTTGCCTTTGGCGACATTGA
DFNRKGGMKQMGKALAGPMVLVM
CGACTTCAACCGCAAGGGCGCATGAAGCAGATGGGCAAGTCATG
SIWDDHASNMLWLDSTFPVDAAG
70
140
210
280
350
420
490
560
630
700
770
840
910
980
1050
1120
1190
1260
1330
1400
1470
1540
TCCATCTGGGATGACCACGCCTCCAACATGCTCTGGCTCGACTCGACCTTCCCTGTCGATGCCGCTGGCA
1610

A.cellulase gene
of
Humicola grisea
2573
uuu
1
UUC
17
UUA
0
Leu
UUG
0
KPGAERGACPTTSGVPAEVEAEAP
AGCCCGGCGCCGAGCGCGGTGCCTGCCCGACCACCTCGGGCCCC
1680
ucu
4
UAU
4
UGU
0
UCC 15
Tyr
UAC
13
cys UGC
24
-
UAA 1
-
UGA
0
Ser
UCA
0
UCG
10
-
UAG
0
Trp UGG
11
NSNVVFSNIRFGPIGSTVAGLPG
CAACAGCAACGTCGTCTTCTCCAACATCC~~TCGGCCCCCAT~~CGACCGTT~T~TCTCCCC~C
1750
cuu
1
CUC 16
Leu
CUA
0
CUG
5
AGNGGNNGGNPPPPTTTTSSAPA
GCGGGCAACGGCGGCAACAACG~~CAACCCCCCGCCCCCCCACCACCACCACCTC~CGGCTCCGGCCA
1820
CCU
6
CAU
0
CGU
3
Pro
CCC
15
His
CAC
6
Arg
E::
1i
CGG
0
Gln
Ett
2:
CCA
0
CCG
4
TTTTASAGPKAGRWQQCGGIGFTG
CCACCACCACCGCCAGCGCTGGCCCCCAAGGCTGGCCGCT~A~AGTGC~~GGC~TCGGCTTCACTGG
1890
AUU
4
Ile AUC
18
AUA
0
Met AUG
11
PTQCEEPYICTKLNDWYSQCL
CCCGACCCAGTGCGAGGAGCCCTACATTTGCACCAAGCTCAACGACT~TACTCTCAGTGCCTGtaaatt
1960
ACU
8
AAU
1
AGU
1
ACC
44
AAC
32
Ser
AGC
8
ACA
0
AAA
0
AGA
0
ACG
1
Lys
AAG
24
Arg
AGG
1
ctgagtcgctgactcgacgatcacggccggtttttgcatgaaaggaaacaaacgaccgcgataaaaatgg
2030
GUU
3
GUC
18
GUA
0
GUG
3
agggtaatgagatgtc
GAU 15 GGU
13
GCU
11
GCC
32
Asp
GAC 18 GGC 50
Ala GCA
0
GAA
1
G1y
GGA
0
GCG
3
G1u
GAG
19
GGG
0
Fig.
3.
The determined DNA sequence of the
H.
griseu
cbh-Z
gene, and the translation
of
the exons. Also indicated,
by
underlining, are
a
possible TATA
box
(193-199),
the internal
SulI
site
from
which sequencing commenced
(771-776),
and the consensus
5',
3'
and
internal sequence features of the intron
(732-734; 789-791).
Table
I.
Codon usage in the cbh-1 gene
of
H.
grisea
give no amino acid sequence equivalent to any part of the
sequence in the other species, and in fact would result in
chain termination at an in-phase UAA codon.
Codon usage in the
H.
grisea
cbh-1
gene is shown in
Table
1.
It shows a marked inequality in frequency of
bases in the third position of the codon, with a strong
preference for uridine. For those amino acids for which it
is necessary to have a purine base in the third position of
the codon, there is a very strong preference for guanine.
Although codon usage in nuclear genes of filamentous
fungi in general is far from random (Kinghorn, 1988), the
bias in this gene (Table
1)
is probably more extreme than
in any other to date, with third-position bases being
337
C,
11
1
G,
75
U
but only
2
A.
This bias against adenine in
the third position is exceptional.
By comparison with other cellulases (Knowles
et
al.,
1987),
the sequence of the
H.
grisea
cbh-1
product can be
divided into domains, with a signal sequence from
residues
1
to 18, an exoglucanase catalytic domain (19-
456),
a 'hinge'
(457-487),
and a cellulose-binding domain
(488-525)
(Fig.
4).
The
H.
grisea
cbh-1
sequence was
analysed using standard methods to determine hydro-
pathy and to make a secondary structure prediction (Fig.
4).
The polypeptide has a hydrophobic N-terminal signal
sequence as expected, but the remainder of the sequence
is predominantly hydrophilic. Predicted a-helix forms
the entire signal sequence, plus several short stretches in
the catalytic domain, but is entirely absent from the
hinge and cellulose-binding domain. Interspersed short
blocks of P-sheet and turn coil form the major part of the

2574
M.
de
0.
Azevedo and others
Sequence
1
100
200
300
400
500
I
I
I
I
I
I
S
Catalytic
HB
(a)
Domains
I1
I
1
I
V
Fig.
4.
Structural features of the
cbh-1
gene product.
(a)
The domain structure, indicating the export signal sequence
(S),
the catalytic
domain, the hinge sequence
(H),
and the cellulosobinding domain
(B).
(b)
Hydropathy plot, prepared by the methods
of
Kyte
8c
Doolittle
(1982).
Above
the horizontal line is hydrophobic, and
below
is hydrophilic.
(c)
Secondary structure plot indicating the relation
to
the catalytic, hinge and cellulose-binding
domains
of the enzyme in
(a).
0,
predicted a-helix;
m,
jhheet;
-,
turn
coil.
Table
2.
Overall
and
domain sequence
homology
between the
cbh-1
gene
of
H.
grisea
and
its
equimlents
in
P.
chrysospri'
and
T.
reesei,
pius
the
related eg-l endoglucanase
of
T.
reesei
The
signal
sequence
has
been
omitted from
this
table.
Domain
:
Gene Catalytic Hinge Binding Total
T.
mesei
cbh-1
250
-
61%
413
17
30
-
57% 20
38
-
53% 287
48
1
-
60%
273
-
56%
21
37
489
-
57%
25
1
1
29
423
-
3%
P.
chrysosporium
cbh-1
-
59%
T.
reesei eg-1
25
1
360
44%
6
28
-
21%
17
38
-
45% 273
426
-
43%
catalytic domain, and are the only structures predicted in
the hinge and cellulose-binding domain. This same
general arrangement of domains is also found in
cbh-1
in
T.
reesei
and
P.
chrysosporium
and in the
T.
reesei eg-l
gene product. In the products of other cellulase complex
genes in
T.
reesei,
the cellulose-binding domain is N-
terminal, and the catalytic domain C-terminal (Knowles
et al.,
1987). Both patterns are found in bacterial
cellulases. The catalytic
domain-hinge-substrate-bind-
ing domain structure is found in certain other polysac-
charide-digesting enzymes, e.g. amylases, and the hinge
may similarly be rich in residues such as threonine, e.g.
glucoamylase
of
Aspergillus niger
(near the C-terminus)
or
Saccharomyces cerevisiae
(near the N-terminus)
(Svensson
et al.,
1986; Yamashita
et al.,
1985). In these
examples also, the hinge is threonine-rich, but has less
sequence homology to the
cbh-1
genes than they do
between themselves.
The optimal alignment of the amino acid sequence of
H.
grisea cbh-1
with its equivalents from
T.
reesei
and
P.
chrysosporiurn
was derived by using the program
MANA-
LIGN
from the
OWL
Database software, running on a
VAX/VMS
computer, and is shown in Fig.
5.
This figure
also indicates the major domains within the sequence
:
N-terminal signal, hinge, and substrate-binding domain,
with the long catalytic domain between the signal and
the hinge.
A
breakdown of the homology for catalytic,
hinge and cellulose-binding domains is shown in Table
2.
In the hinge region, the
H.
grisea
sequence resembles that
of
T.
reesei cbh-1
in being rich in proline and threonine
residues, although these both differ markedly from the
P.
chrysosporium ebh-1
hinge, which is rich in serine
residues. Except for the hinge region, however, the three
sequences are more or less equally related, with about
60% homology. This perhaps suggests that the proline/
threonine-rich hinge of
H.
grisea
and
T.
reesei
is typical
of
Fig.
5.
Optimal alignment
of
the
H.
grisea
cbh-1
amino-acid sequence with those of the equivalent
cbh-l
sequences
of
P.
chrysosporium
and
T.
reesei,
and also
of
the related
eg-1
(endoglucanase) sequence
of
T.
reesei.
The signal, hinge and substrate-binding domains are
indicated, and the long catalytic domain is between the signal and the hinge. Asterisks and vertical lines denote identical residue in
adjacent sequence, and identical residue in non-adjacent sequence, respectively.

A
cellulase gene
of
Humicola grisea
2575
Signal
Humicola cbh-1
I I
MRTAKFATLAALVASAAAQQACSLTTERHPSLSWNKCT-AGGQCQTVQAS
* *
**:;
;*
;
**
*
;
***
***
;
1
I
I
I,*
Phanerochaete cbh-1
MFRTATL~AFTMAAMvF~OQllGTNTARSHPALTSQKC~~GCS-NLNTK
Trichoderma cbh-I
KYRKLAVISAFLATARAQSACTLQSETHPPLTWQKCS-SGGTC-TQQTG
**
*;;
*;;:*
**;**
;
**;;***:
*
*;
Trichoderma eg-I
MAPS~TLTTAILAIARL~AAQQPGTSTPEVHPKLTTM<CTKSGGCV-AQDTS
*
*I
I,,
'*I I,
I
I
I
**;**;***;;***;;
;
;*
ITLDSNWRWTDHQVSGSTNCYTGNKWI-TSICTDAKSCAHNCCVDGADYTSTYGITTNGDSLSLKFVTKGQHST-
*
**
****;
*
**;*******
*
*
*
*
*
**
**;
;*****;*****;
*
**;*
***
IVLDANWRWL-HSTSGYTNCYTGNQWDAT-LCPDGKTCAANCALSGADYTGTYGITASGSSLKLNFVTGS-----
SVIDANWRWT-HATNSSTNCYDGNTWSST-LCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQK--
VVLDWNYRWM-HDANY--NSCTVNGGVNTTLCPDEATCGKNCFIEGVDYAAS-GVTTSGSSLTMNQYMPSSSGGY
*;******;
*
*
;****;**
* *
****
***
**;*;**;*
;***
*;**;**;
;***
*
*;*
*
**
*
;*
*
;
* *
****
**
***
*
;**
******;**
;
*
-NVGSRTYLMDGEDKYQTFELLGNEFTFDVDVSNIGCGLNGALYFVSMDADGGLSRYPGNKAG~YGTGYCDAQC
*****
***
**
*
**;;*******;**
********;;:******
;
**
*************;**
-NVGSRYYLMADDTHYQMFQLLNQEFTFDVDMSNLPCGLNGALYLS~DADGGM~YPTNKAG~YGTGYCDSQC
***
*
****
**
**;*
**;
;**
****;*
**********;;;******
;*****
**************
-NVGARLYIlVIASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAG~YGTGYCDSQC
*
*I**
**
*
I
*
*:*
*****
*
****
** **:;
**
**
*
*****
**
****;**
SSVSPRLYLLDSDGEYVMLKLNGQELSFDVDLSALPCGENGSLYLSQMDENGGANQY--NTAGANYGSGYCDAQC
PRDIKFINGEANIEGWTGSTNDPNAGAGRYGTCCSEMDIWEANNMATAFTPHPCTIIGQSRCEGDSCGGTYSNER
************
***
;;;
;
*
;
****;*********
*
*
******
;*
**;*;
*;:;;;
;
PRDIKFINGEANVEGWNATSANAGTGNY--GTCCTEMDIWEANNDAAAYTPHPCTTNAQTRCSGSDCTRDT----
***
*****
I*****
;*
**;**
;
*
**;********
;
*;*******
;*
*;*;
*;;;;;
;
PRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTV~EICEGDGCGGTYSDNR
*
* *
'*
*
**
****;*
**
;
*****
**
;
p-----------
VQTWRNGTLNTSHQ----GFCCNEMDILEGNSRANALTPHSCTATA-----------------
YAGVCDPDGCDFNSYRQGNKTFYGKGM--TVHTTKKITVVTQFLKDANGDL-----GEI~FYVQDGKIIPNSES
I
*
**
*;
;****;* *I;**;***
**
*;*;
******
;
***
** *
***
**
*;**
--GLCDADCGDFNSFRMGM/TFLGKGL--TVDTSKPFTVVTQFIT--NGDTSAGTLTEIRRLYVQNGKVIQNSSV
I
*
**
*;;*
*
;*
*;
*;*
*
*;**;*
;***a**
*
;;*
;
;
; ;
*
*****
;*
YGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFET--SGAIN--------
RYYVQNGGTFQQPNA
**
**
***
*
;*
****
*;**;*
;*
***
*;
;*
;;
; ;
;
* *
***
;;
----
CDSAGCGFNPYGSGYKSYYGPGD--TVDTSKTFT
I I
TQFNTD-NGSPS-GNLVS
I
TRKYQQNGVD IPSAQ-
TIPGVE-GNSITQDWCDRQKVAFGDIDDFNRKGAMKQMGKAFAGPMVL~SIWDDHASNMLWLDSTFPV-DAAGK
KIPGIDPVNSIDTNFCSQQKTAFGDTNYFAQHGGLKQVGEALRTGMVLALSIWDDYAANMLWLDSNYPTNKDPST
;*
;
;*;
;
*
**
*
;***
*
::*;
;****;;*:****
********;****
**
EL-GSYSGNELNDDYCTAEEAEFGGSS-FSDKGGLTQFKKSLWDDYYANMLWLDSTYPTNETSST
a*
; ;
--PGGDTISSCPSASAY---------------
GGLATMGKALSSGMVLVFSIWNDNSQYMNWLDSGNAGPC----
***
;***
*;
*
**
****
*
;*
**;*;*
;
***;;*****
*
*******;
ff
***
*I**l*
*****
*;*
*
*
****
I,
I
Hinge
PGAERGACPTTSGVPAEVEAEAPNSNVDFSN
I
RFGP
I
,I,,*III,I I
,,I,
,,,It
I
**;
**
*
*******
;**
**
*
****
**;;;;*
*
1
111
VII,
PGVARGTCATTSGVPAQIEAQSPNAYVVFSNIKFGDLNTT~~GTVSSSSVSSSHSSTSTSSSHSSSSTPPTQPT-
**;
**
*
*;******;*;*****
*;*******;;;;*
*
:
;;;
;;;;;
;;;;
;;;;;
;
PGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGST-----GNPSGGNPPGGNRG--TTTT~PATTTGSS
*
;
*
*
1
'
**
*;****
*;****
ff
**
;;
;
****
**
**
__--___-
SSTEGNPSNILANNPNTHVVFSNIRWGDIGST-----TNSTAPPPPPASSTTFS-TTRRSSTTS-SS
I
III
I41
I,
Substrate-binding
I
I
AGPKAGRWQQCGGIGFTGPTQCEEPYICYKLNDWYSQCL
*;
*
******
**;*
*
**
*
**;
****;
-GVTVPQWGQCGGIGYTGSTTCASPYTCHVLNPDYSQCY
*;*
:********
*;*;***
**
****
****;
PGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL
*
**
*:**********
;
;*;******
;*;******
PSCTQTHWGQCGGIGYSGCKTCTSGTTCQYSNDYYSQCL

2516
M.
de
0.
Azevedo
and
others
this gene in ascomycetes, whereas the serine-rich hinge
of
P.
chrysosporium
is a basidiomycete characteristic.
This work was supported in part by CNPq, Brazil.
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