Download Analysis of the glycoside hydrolase family 8 catalytic core in

International Journal of Computational Bioinformatics
and In Silico Modeling
Vol. 3, No. 1 (2014): 315-320
Research Article
Open Access
ISSN: 2320-0634
Analysis of the glycoside hydrolase family 8 catalytic
core in cellulase-chitosanases from Bacillus species
Niveditha Prakash and Shubha Gopal*
Department of studies in Microbiology, University of Mysore, Manasagangothri, Mysore, Karnataka, India
*Corresponding author: Shubha Gopal; e-mail: [email protected]
Received: 01 December 2013
Accepted: 12 December 2013 Online: 03 January 2014
ABSTRACT
The glycoside hydrolase family 8 (GH-8) consists of bifunctional cellulase-chitosanases many of which are
produced by species of Bacillus. Chitosanolytic enzymes can be useful in producing low molecular weight
chitooligosaccharides which have several applications. In addition, a bifunctional enzyme would be more
beneficial than the use of two individual enzymes in the production of chitooligosaccharides and in the
degradation of the complex cellulose, chitin and chitosan rich biomass that occurs in nature. The crystal structure
of a previously determined GH-8 chitosanase has revealed that the catalytic site is constructed on a scaffold of a
double α6/α6 barrel which consists of six helix-loop-helix motifs. Bifunctional cellulase-chitosanases from
Bacillus species isolated hitherto were analysed and were found to have the double α6/α6 barrel as previously
predicted. The signature pattern of glycoside hydrolase family 8 (GH-8) was determined for the sequences. Some
of the protein sequences among them were modelled and their closest structural analogs were determined. The
structures were found to be related to endoglucanases, xylanases, epimerases and also terpenoid cyclases all of
which have the double α6/α6 barrel architecture. This study provides a detailed insight into the structure of
cellulase-chitosanase catalytic core and identifies related enzymes with similar catalytic core thereby signifying a
possible evolutionary relationship.
Keywords:
Glycoside hydrolase family 8; Bacillus; six hairpin glycosidases; chitosanase-cellulases;
homology modelling.
INTRODUCTION
Chitosan is hydrolysed by chitosanase [EC 3.2.1.132],
an enzyme that catalyses the hydrolysis of glycosidic
bonds of chitosan, the deacetylated derivative of chitin
[1]. Chitosanases are classified under glycoside
hydrolases, a diverse group of enzymes containing 93
families [2]. Bifunctional enzymes with cellulolytic and
chitosanolytic activity mostly belong to glycoside
hydrolase family 8 (GH-8) [3]. GH-8 is a chitosanase
family which also has cellulase, xylanase, lichenase etc.
Most of the bifunctional cellulase-chitosanases,
particularly the ones produced by Bacillus species
belong to glycoside hydrolase family 8. Their primary
sequences show high homology with other glucanases
of GH-8; xylanases, endoglucanases, lichenases and a
few others [3]. Previously resolved crystal structure of
GH-8 chitosanases catalytic domain from Bacillus sp.
K17 by Adachi et al. [4] has revealed that the molecule
consists of six α-helices forming a central barrel,
http://bioinfo.aizeonpublishers.net/content/2014/1/bioinfo315-320.pdf
surrounded by six other α-helices. Six repetitions of
this helix-loop-helix motif form a typical α6/α6 double
barrel structure. The structures have long loops with
short β-strands and 310 helices protrude from the end
of the outer helices and fold towards the inner helices
forming a long cleft on the top of the double barrel
suggestive of substrate binding pockets. On the bottom
of the double barrel structure, short loops link the
helices [4]. GH-8 chitosanases having the typical α6/α6
double barrel structure are called six hairpin
glycosidases. The six-hairpin glycoside domain contains
about seven alpha-hairpins arranged in closed circular
assortment. In the present study, bifunctional cellulasechitosanases that have been previously reported are
structurally examined for presence of the six hairpin
glycoside domain. The protein structures are generated
using online homology modelling servers. GH-8
signature motif is determined for the sequences, their
active site residue is predicted and their closest
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structural analogs are detected. The presence of the
α6/α6 double barrel architecture in proteins from
different families although their active-site architecture
may be different suggests the stability of the α6/α6
motif and the probable evolutionary relationship
between different glycosyl hydrolases [5].
MATERIALS AND METHODS
Analysis of cellulase-chitosanase sequences from
Bacillus species
Previously reported protein sequences of Bacillus
species showing cellulase and chitosanase activities
were
retrieved
from
NCBI
[www.ncbi.nlm.nih.gov/protein] with the following
accession numbers: Chitosanase from Bacillus cereus
H1 (Q849S1) [6], Bacillus sp. strain KCTC 0377BP
(QALZ1) [7] and cellulase-chitosanases from Bacillus
thuringiensis subspecies (AB061887, AB061888,
AB061889,
AB061891,
AB061892,
AB061893,
AB061894 and AB061895) [8].
Multiple sequence alignment and analysis of the
glycosyl hydrolases family 8 signature motif
The protein sequences were aligned using Clustal
Omega
set
at
default
parameters
[www.ebi.ac.uk/Tools/msa/clustalo] [9-10].
The
Multiple sequence alignment output from Clustal
Omega
was
obtained
using
Jalview
[http://www.jalview.org/] [11]. The protein sequences
were
analysed
with
Prosite
[http://prosite.expasy.org/scanprosite/] [12] and the
signature motifs of the enzymes were obtained. The
position of the signature motif in the multiple sequence
alignment was detected. Sequence logo was generated
using Weblogo [http://weblogo.berkeley.edu/logo.cgi]
[13].
Homology modelling and identification of
structural homologs
Chitosanase-cellulase protein sequences of the above
mentioned protein sequences were retrieved from
NCBI [www.ncbi.nlm.nih.gov/protein]. Chitosanase
from Bacillus cereus H1 (Q849S1) [6], Bacillus sp. strain
KCTC 0377BP (QALZ1) [7] and one from Bacillus
thuringiensis
subspeciesAB061887
(Bacillus
thuringiensis serovar alesti) [8] were used for the
homology modelling. Homology modelling was done
using the Swiss model workspace in the automatic
modelling mode [http://swissmodel.expasy.org/] [1416]. The template used for homology modelling was
1V5CA. Protein data bank (PDB) homologs were
determined for the sequences using the COFACTOR
server
[zhanglab.ccmb.med.umich.edu/COFACTOR/]
[17].
RESULTS AND DISCUSSION
Analysis of cellulase-chitosanase sequences from
Bacillus species
Chitosanolytic enzymes are gaining importance as they
can produce low molecular weight compounds
including chitooligomers with high yield and low
pollution. They have numerous applications in
http://bioinfo.aizeonpublishers.net/content/2014/1/bioinfo315-320.pdf
biomedical, pharmaceutical, agricultural and food fields
[18]. The following previously isolated cellulasechitosanases whose sequences were deposited in
Genbank were selected for the in silico analysis.
Chitosanase from Bacillus cereus H1, which showed
CMC and glycol chitosan activity (Accession number
Q849S1) [6] and soil bacterium Bacillus sp. strain KCTC
0377BP isolated by Choi et al. [7] (Accession number
QALZ1). Lee et al. In 2007 screened several Bacillus
thuringiensis subspecies which produced bifunctional
cellulase-chitosanases (Accession numbers: AB061887,
AB061888,
AB061889,
AB061891,
AB061892,
AB061893, AB061894, AB061895) [8].
Multiple sequence alignment and study of the GH-8
signature motif
The retrieved protein sequences were aligned with
Clustal Omega [www.ebi.ac.uk/Tools/msa/clustalo] [910] and the output of the multiple sequence alignment
was obtained from Jalview [http://www.jalview.org/]
[11]. The multiple sequence alignment output which
contains the signature motif is shown in figure 1. The
signature motif of the GH-8 family is a stretch of about
20 residues. The signature motif sequence has of two
conserved aspartates. The first aspartate is believed to
be a nucleophile in the catalytic mechanism [5]. The
following consensus pattern was obtained:
A-[ST]-D-[AG]-D-x(2)-[IM]-A-x-[SA]-[LIVM][LIVMG]-x-A-x(3)-[FW], where the first D is the active
site residue.
The signature motif was generated from Prosite
[http://prosite.expasy.org/scanprosite/] [12] and the
sequence logo was created. Sequence logo is a graphical
representation of multiple sequence alignments. It has
stacks of letters which are colour coded and represent
amino acids at consecutive positions. Sequence logos
are more precise than the consensus sequence. The
total height of the logo is based on the degree of
conservation of the sequence. The height of the amino
acids in the stack pertains to the relative frequency of
the amino acid at that position. The logos are read from
the top of the stacks [20]. The colouring of the amino
acids is based on their chemical properties. Polar amino
acids have been coloured green (T- threonine, GGlycine, Y- Tyrosine, S- Serine), Basic amino acids are
coloured blue (K- Lysine, H- Histidine), Acidic amino
acids are coloured red (D- Aspartic acid) and
hydrophobic amino acids (A- Alanine, L- Leucine and IIsoleucine)
are
coloured
black
[http://weblogo.berkeley.edu/info.html#color_sym].
Sequence logo was created using the online tool
Weblogo [http://weblogo.berkeley.edu/logo.cgi] [13].
Sequence logo of the multiple sequence alignment is
shown in figure 2.
Homology modelling of cellulase-chitosanase
sequences
Homology modelling for the cellulase-chitosanase
sequences was done in the automatic modelling mode
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using
the
Swiss
model
server
[http://swissmodel.expasy.org/] [14-16]. Homology
modelling revealed that all the selected cellulasechitosanases sequences had the α6/α6 domain
architecture which is typical of GH-8 members [5]. The
homology models of the cellulase-chitosanase
sequences with their signature motifs and catalytic
domains are shown in figures 2, 3 and 4. Front view of
the structures show the presence of the double barrel
α6/α6 motif while the side views show the catalytic
cleft and the N and C termini of the proteins. Signature
motif of the proteins was seen in sequences 181-199.
The first aspartate of the signature motif (Asp 183) was
believed to be the active residue of the catalytic
mechanism. The homology models were refined by
using Modrefiner (http://zhanglab.ccmb.med.umich.
edu/ModRefiner/) [21]. The quality of the structures
that were modelled was verified with PROCHECK
obtained from PDBsum. [http://www.ebi.ac.uk/
thornton-srv/databases/cgi-bin/pdbsum/] [21].
Figure 1. A part of the multiple sequence alignment showing the GH-8 signature motif. The signature motif is coloured purple.
Figure 2. Sequence logo of the GH-8 signature motif. Sequence logo
[http://weblogo.berkeley.edu/logo.cgi]. Signature motif is found in the position 181-199.
(a)
was
generated
from
Weblogo
(b)
Table 1. Plot statistics for Bacillus cereus H1 chitosanase
Residues in most favoured regions [A,B,L]
Residues in additional allowed regions [a,b,l,p]
Residues in generously allowed regions
[~a,~b,~l,~p]
Residues in disallowed regions
300
39
0
88.2%
11.5%
0.0%
1
0.3%
Number of non-glycine and non-proline
residues
Number of end-residues (excl. Gly and Pro)
Number of glycine residues (shown as
triangles)
Number of proline residues
340
100.0%
Total number of residues
386
1
28
17
(c)
Figure 3. Front view (a), side view (b), Ramachandran plot (c) and plot statistics for chitosanase from Bacillus cereus H1
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(d)
(e)
Table 2. Plot statistics for Bacillus sp. strain KCTC 0377BP
Residues in most favoured regions [A,B,L]
Residues in additional allowed regions [a,b,l,p]
305
89.7%
34
10%
0
1
0.0%
0.3%
Number of non-glycine and non-proline residues
Number of end-residues (excl. Gly and Pro)
340
1
100%
Number of glycine residues (shown as triangles)
Number of proline residues
28
17
Residues in generously allowed regions [~a,~b,~l,~p]
Residues in disallowed regions
Total number of residues
386
(f)
Figure 4. Front view (d), side view (e), Ramachandran plot (f) and plot statistics for chitosanase Bacillus sp. strain KCTC 0377BP
(g)
(h)
Table 3. Plot statistics for chitosanase, Bacillus thuringiensis serovar
alesti
Residues in most favoured regions [A,B,L]
304
89.4%
Residues in additional allowed regions [a,b,l,p]
34
10.0%
1
0.3%
1
0.3%
Number of non-glycine and non-proline residues
Number of end-residues (excl. Gly and Pro)
340
1
100.0%
Number of glycine residues (shown as triangles)
Number of proline residues
28
17
Residues in generously allowed regions
[~a,~b,~l,~p]
Residues in disallowed regions
Total number of residues
386
(i)
Figure 5. Front view (g), side view (h), Ramachandran plot (i) and plot statistics for chitosanase Bacillus
thuringiensis serovar alesti
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Identification of structural homologs
In order to detect the occurrence of proteins with
structural similarity, the COFACTOR server was used
[zhanglab.ccmb.med.umich.edu/COFACTOR/] [16]. The
top 10 identified structural analogs from PDB were
retrieved and tabulated as shown in table 4. All the
structures share the common α6/α6 domain. This
clearly shows that the cellulase-chitosanases from the
selected Bacillus species are structurally related to
cellulases, xylanases, endoglucanases, epimerases and
terpenoid cyclases from different organisms.
Table 4. Top 10 structural analogs of chitosanases from Bacillus cereus H1, Bacillus sp. strain KCTC 0377BP and Bacillus
thuringiensis serovar alesti as identified from the COFACTOR server
Sl.
No.
Chitosanase, Bacillus cereus H1
Chitosanase, Bacillus sp. strain KCTC 0377BP
B.thuringiensis
serovar
cellulase-chitosanase
structural
analogs
name of the enzyme
structural
analogs
structural
analogs
Name of the enzyme
1
1v5cA
Chitosanase
1l2aA
Processive endocellulase CelF
1l2aA
2
1cemA
Cellulase CelA
2afaB
3
2b4fA
Endo-1,4-β-xylanase
3gt5A
N-acylglucosamine
(AGE)
N-acylglucosamine
(AGE)
3gt5A
Processive
endocellulase CelF
N-acylglucosamine
2-epimerase (AGE)
N-acylglucosamine
2-epimerase (AGE)
name of the enzyme
2-epimerase
2afaB
2-epimerase
alesti
4
2drrA
Xylanase Y
3qxqA
Endoglucanase
3qxqA
Endoglucanase
5
4fusA
Endoglucanase F
1wzzA
Probable endoglucanase (cmcAX)
1wzzA
6
4el8A
Endoglucanase F
1fp3B
N-acylglucosamine
(AGE)
2-epimerase
1fp3B
Probable
endoglucanase
(cmcAX)
N-acylglucosamine
2-epimerase (AGE)
7
2zblB
N-acylglucosamine
2-epimerase (AGE)
3vw5A
N-acylglucosamine
(AGE)
2-epimerase
3vw5A
N-acylglucosamine
2-epimerase (AGE)
8
2rgkC
N-acylglucosamine
2-epimerase (AGE)
2zzrA
Glycosyl hydrolase
2zzrA
Glycosyl hydrolase
9
1l2aA
2fuzA
Glycosyl hydrolase
2fuzA
Glycosyl hydrolase
10
3gt5A
Processive endocellulase
CelF
N-acylglucosamine
2-epimerase (AGE)
1w6jA
Terpenoid cyclase
1w6kA
Terpenoid cyclase
CONCLUSION
GH-8 is a diverse group of multifunctional enzymes
having chitosanases, cellulases, xylanases, lichenases
etc. Cellulase-chitosanases belonging to this family are
gaining importance as they can produce low molecular
weight chitooligomers which are commercially
important for the pharmaceutical, agricultural and food
industries. Bacillus species which are common soil
inhabitants are known to produce cellulasechitosanases [3]. Several cellulase-chitosanases have
been isolated so far, but their structural details are not
clearly understood. The protein sequences of cellulasechitosanases were shown to have a signature motif of
about 20 amino acids were the first aspartate is
believed to be the nucleophile in the catalytic
mechanism. All GH-8 proteins analysed here were
found to have the α6/α6 double barrel structure.
Significant structural similarity was seen among
cellulases, xylanases, epimerses, endoglucanases and
terpenoid cyclases from different organisms. This
probably indicates the evolutionary relationship among
different glycoside hydrolases having the α6/α6 double
barrel catalytic core and the evolutionary stability of
the α6/α6 motif among the glycoside hydrolases of
several species [5]. An attempt has been made in this
study to understand the structures of these beneficial
http://bioinfo.aizeonpublishers.net/content/2014/1/bioinfo315-320.pdf
enzymes and also establish their similarity with
enzymes having related structures and function.
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