Molecular Docking and Site-directed Mutagenesis of a Bacillus

Int. J. Biol. Sci. 2015, Vol. 11
Ivyspring
International Publisher
304
International Journal of Biological Sciences
Research Paper
2015; 11(3): 304-315. doi: 10.7150/ijbs.10632
Molecular Docking and Site-directed Mutagenesis of a
Bacillus thuringiensis Chitinase to Improve Chitinolytic,
Synergistic Lepidopteran-larvicidal and Nematicidal
Activities
Hong Ni1,2, Siquan Zeng2, Xu Qin1, Xiaowen Sun2, Shan Zhang2, Xiuyun Zhao1, Ziniu Yu1, Lin Li1,
1.
2.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Faculty of Life Science, Hubei University, Wuhan
430062, Hubei, China
 Corresponding author: Mail address: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan
430070, and China. Telephone: +86-27-87286952. Fax: +86-27-87280670. E-mail: [email protected]
© 2015 Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Please see http://ivyspring.com/terms for terms and conditions.
Received: 2014.09.24; Accepted: 2014.12.24; Published: 2015.01.30
Abstract
Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low
enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with
enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and
substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602
(Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of
Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using
di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the
docking area for the site-directed mutagenesis experiments and expression in Escherichia coli.
Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase
in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to
wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations
against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several
plant-pathogenic fungi were observed.
Key words: Chitinase; Bacillus thuringiensis; Homology modeling; Molecular docking; Site-directed mutagenesis; Synergistic activity.
INTRODUCTION
Chitinases (E.C.3.2.1.14) comprise a family of
extracellular cell-wall hydrolases that hydrolyze the
β-1,4-glycosidic bond of chitin to form the monomer
N-acetyl-D-glucosamine. Chitinases are found in a
wide range of organisms, including bacteria, plants,
fungi, insects, and crustaceans [1]. Bacterial chitinases
have potential applications in the suppression of
plant-pathogenic fungi and nematodes, the control of
various pests, and the recycling of chitin to generate
carbon and nitrogen sources in ecosystems [2, 3]. Of
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Int. J. Biol. Sci. 2015, Vol. 11
the
various
chitinase-producing
bacteria,
Gram-positive Bacillus species are of special interest
due to their ability to form endospores that are resistant to heat and other adverse environments, an
advantageous characteristic for agricultural applications [3].
Bacillus thuringiensis is an important insecticidal
bacterium that produces insecticidal crystal proteins
(ICPs) during sporulation. A variety of B. thuringiensis
strains also produce chitinases [1, 4-6]. Because the
peritrophic membrane of the insect midgut is composed of a chitinous matrix, the presence of chitinases
in B. thuringiensis formulations may increase the uptake of ICPs into epithelial cells and enhance insecticidal activity [7, 8]. Thus, this bacterium is an attractive host for the development of a biocontrol agent
with enhanced activity through increasing the expression of chitinases in vivo or through creating a
formulation of ICPs and chitinases in vitro. Previous
studies have demonstrated that the addition of bacterial chitinases enhances insecticidal effects of B. thuringiensis ICP preparations against pest larvae [9, 10].
Unfortunately, the endogenous chitinase activity of
wild-type B. thuringiensis strains is relatively low [1,
6], and improving the chitinolytic activity of B. thuringiensis-derived chitinases would facilitate the development of B. thuringiensis composite formulations
[11].
Protein engineering via site-directed mutagenesis is an essential tool for creating a mutant enzyme
with altered or novel properties. To improve chitinase
activity, site-directed mutagenesis of the Vibrio harveyi
chitinase VhChiA, in which amino acid Y435 was replaced with A435, resulted in a mutant enzyme with
enhanced binding and catalytic efficiency [12]. The
W397F mutant of Vibrio carchariae chitinase A enhanced the hydrolysis of the pNP substrate by 142%,
with activity toward colloidal chitin similar to that of
the wild-type enzyme [13], while the Y245W mutation
at the predicted exterior site of the catalytic cleft enhanced the hydrolysis of crystalline alpha-chitin and
colloidal chitin [14]. In Serratia marcescens, the ChiA
D313N and ChiB D142N mutants exhibited strongly
enhanced transglycosylating activity [15]. These results suggest that structural mutations of active site
residues may enhance the hydrolytic activity of chitinases.
B. thuringiensis chitinases structurally consist of a
signal peptide, a catalytic domain, a fibronectin
type-III-like domain (Fn3D), and a chitin-binding
domain [16, 17]. Based on amino acid sequence similarity, these chitinases are grouped into family 18
glycosyl hydrolases [18, 19], which feature a
three-dimensional structure in which the catalytic
domain is located in a TIM barrel structure composed
305
of eight α-helices and eight β-strands [20, 21]. This
conserved molecular structure enables the analysis of
amino acid residues critical for enzymatic reactivity
using molecular docking and site-directed mutagenesis techniques.
No effort has yet been made to improve the catalytic activity of a B. thuringiensis chitinase through
molecular docking and site-directed mutagenesis
techniques. In the present study, based on the analysis
of the three-dimensional protein structure of the B.
thuringiensis chitinase Chi9602 by homology modeling
and molecular docking analysis, the impact of 10
amino acid codons on enzymatic activity was analyzed by site-directed mutagenesis and specific chitinolytic activity assays, and three enzyme variants
with enhanced activity were identified. The ChiW50A
mutation increased activity by more than 60%, and
the effects of pH, temperature, and several metal ions
on the activity of the ChiW50A mutant and wild-type
Chi9602 and their catalytic kinetic constants were
compared. The synergistic pest-controlling effects of
ChiW50A on B. thuringiensis spore-ICP preparations
against Helicoverpa armigera and Caenorhabditis elegans
larvae were also examined.
MATERIALS AND METHODS
Bacterial, fungal and C. elegans strains; genes;
plasmids; and culture conditions
E. coli DH5α cells (TaKaRa Bio, Inc.) were used to
construct various recombinant plasmids. E. coli TOP10
[F-, mcrA Δ (mrr-hsdRMS-mcrBC) Φ80lacZΔM15
ΔlacX74 recA1 araD139 (ara-leu)7697galU galK rpsL
endA1 nupG] (Invitrogen) was used as the host strain
for the expression and purification of target proteins.
The fungal strains Sclerotinia sclerotiorum FB014, Physalospora piricola FB016, Fusarium oxysporum FB012,
Fulvia fulva FB009, Botrytis cinerea FB007 and Rhizoctonia solani FB010 (Microbial Genetic Stock Center,
Wuhan, China) were used as indicators for antifungal
assays. B. thuringiensis wild-type strain YBT-1520
(CCTCC No. M94067, China Center for Type Culture
Collection), which has lepidopteran-larvicidal activity, and wild-type strain YBT-020 (Microbial Genetic
Stock Center, Wuhan, China), which has nematicidal
activity, were used to prepare spore-ICP preparations.
Neonate larvae of a susceptible strain of H. armigera
artificially fed in the laboratory (KeNuo Biotech Co.
Ltd, Wuhan, China) and synchronized fourth-stage
(L4) larvae of C. elegans wild-type strain N2 (Bristol)
were used for toxicity bioassays of the B. thuringiensis
spore-ICP preparations.
The chitinase gene chi (GenBank acc. no.
KF671757.1) was isolated from B. thuringiensis subsp.
tenebrionis wild-type strain YBT-9602 (Microbial Gehttp://www.ijbs.com
Int. J. Biol. Sci. 2015, Vol. 11
netic Stock Center, Wuhan, China). The recombinant
plasmid pMB332 (Supplementary Material: Fig. S1),
which harbors the encoding sequence (codons 35 to
676) of chi and expresses the Chi9602 protein in E. coli,
was used as the DNA template to amplify various
mutated chi genes by polymerase chain reaction
(PCR). The recombinant plasmids pMB581-Y46A,
pMB581-W50A, pMB581-R55A, pMB581-W171A,
pMB581-E211A, pMB581-D287A, pMB581-R343A,
pMB581-D385A,
pMB581-W447A,
and
pMB581-S450A, which harbored single-amino acid
mutations of the chi gene (Fig. S1), were constructed to
express and purify the mutant Chi9602 proteins.
E. coli strains harboring various recombinant
plasmids were grown in Luria−Bertani (LB) medium
containing 100 µg/mL ampicillin (Amp) at 37 °C. B.
thuringiensis strains were cultured in 500-mL Erlenmeyer flasks containing 50 mL of LB broth at 210 rpm
and 37 °C, unless specified otherwise. Fungal strains
were grown in PDA medium (20% potato infusion 100
mL, dextrose 2 g, biological agar 1.5 g) at 28 °C. The C.
elegans N2 strain was cultured using the method previously described by Lewis and Fleming [22].
Homology modeling and molecular docking
The chi sequence was characterized by conducting BLASTN and BLASTP searches of the GenBank
nucleotide and amino acid sequence databases at the
National Center for Biotechnology Information
(NCBI)
server
(http://blast.ncbi.nlm.nih.gov/
Blast.cgi). The conserved domain architectures of
Chi9602 were analyzed using the NCBI online tool
“Conserved Domain Search” (http://www.ncbi.nlm.
nih.gov/Structure/cdd/wrpsb.cgi). The signal peptide
sequence
was
predicted
by
SignalP
(http://www.cbs.dtu.dk/services/SignalP/).
For homology modeling of the Chi9602 catalytic
domain and partial downstream codons (from codon
35 to 459, in brief, Chi960235-459), homologous proteins
were first identified by searching the Protein Data
Bank (PDB) database (http://www.rcsb.org/pdb/
home/home.do). All matched proteins were aligned
based on sequence similarity to Chi960235-459, and the
proteins with similarities greater than 30% were considered candidate reference proteins for homology
modeling. A structural motif (PDB ID: 1ITX) of the
catalytic domain of Bacillus circulans WL-12 chitinase
A1 [23] with 61% sequence identity to Chi960235-459 at
the corresponding codon regions was then selected as
the reference protein.
Homology modeling was performed based on
the 1ITX.1.A reference protein model using the “Easy
modeler 4.0” tool in the software “MODELLER 9.13”.
An optimized Chi960235-459 homology model was
constructed from the three generated models by se-
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lecting the “DOPE profile” options. The Chi960235-459
homology model was validated using the online tool
“Molprobity”
(http://molprobity.biochem.duke.
edu/index.php) and evaluated by the “Ramachandran Plot Analysis” online tool (http://mordred.bioc.
cam.ac.uk/~rapper/rampage.php). Subsequently, a
stereo image of the Chi960235-459 ribbon model illustrating the TIM-barrel structure and a stereo image
illustrating the surface groove structure of the
Chi960235-459 model were generated using “PyMol”
software (DeLano Scientific LLC).
To perform molecular docking of Chi960235-459
and NAG, a di-[N-Acetyl-D(+)-glucosamine] [(NAG)2]
(CID: 446943) molecular ligand was obtained from
“PubChem”
(http://pubchem.ncbi.nlm.nih.gov/).
Protein docking of the Chi960235-459 model with
(NAG)2 as the substrate was conducted using AutoDock 4.2 software (The Scripps Research Institute), in
which the program Autogrid 4.2 was run at a mesh
region with the “Numbers of points in X-, Y-, and
Z-dimensions” of 80, 70, and 80, respectively, “Spacing (Å)” of 0.375, and residue E211 set as the center. A
total of 20 matched docking conformations were generated using E211 as the flexible residue, and the
number of rotatable bonds of (NAG)2 was set as 6/32.
An optimal docking configuration of Chi960235-459 and
(NAG)2 based on these docking conformations was
determined by adjusting the “Binding Energy” to a
minimal value of -8.15 kcal/mol. This docking configuration was then plotted as a 3D image using the
tool PyMol. The docking area of Chi960235-459 and
(NAG)2 featured a substrate-binding, semi-closed
groove channel, and 27 amino acid residues were
identified as pocket residues in the receptor-ligand
groove structure using the Discovery Studio Visualizer tool (Accelrys). From these residues, 10 (Y46,
W50, R55, W171, D287, E211, R343, D385, S450, and
W447) were selected that were predicted to pack the
substrate and form the substrate-binding groove
based on their steric distances from (NAG)2 and their
hydrophilic/hydrophobic nature.
Plasmid construction and expression and purification of chitinases
The oligonucleotide primers used in this study
are listed in Supplementary Material: Table S1. The
recombinant plasmids harboring mutated chi genes
with either a single mutated codon or dual mutated
codons are schematically illustrated in Supplementary
Material: Fig. S1. Briefly, the full-length chi was amplified by PCR from the B. thuringiensis YBT-9602 genome with primers Chi-F (Table S1, BglII site underlined) and Chi-R (Table S1, PstI site underlined). The
PCR-amplified fragment was sequenced before digestion with BglII and PstI. The digested fragment
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Int. J. Biol. Sci. 2015, Vol. 11
307
was then ligated to the BglII/PstI site of the E. coli
expression vector pTrcHis B (Invitrogen), yielding the
recombinant plasmid pMB332. To obtain a
site-directed chi mutant in which the amino acid of
interest was replaced with Ala, three separate PCRs
were performed using the standard SOE (splicing by
overlap extension) method [24]. For example, to construct chiY46A (in which the codon for Tyr46 was replaced with Ala46), the Chi-F (Forward) and Y46A-R
(Reverse) primer pair was used to amplify a fragment
containing the upstream sequences and the mutation
site (from codon 35 to 46), and the second primer pair,
Y46A-F and Chi-R, was used to amplify the mutation
site and the downstream sequences (from codon 46 to
676). The third primer pair, Chi-F and Chi-R, was
used to generate the full-length chi mutant (from codon 35 to 676), with the previously amplified overlapping fragments used as the heteroduplex templates. A similar protocol was used to construct other
mutant chi genes with single-codon mutations. To
construct the double mutant chiW50A/S450A (W50A
and S450A), a similar SOE method was employed,
except the previously constructed chiW50A gene was
used as the DNA template. All final amplified mutant
chi genes were verified by sequencing and ligated into
the BglII/PstI sites of pMB332 to yield the recombinant plasmids listed in Supplementary Material: Fig.
S1.
Transformed E. coli cells expressing wild-type or
mutant Chi9602 were grown in fresh LB broth containing 100 µg/mL Amp and incubated at 37 °C until
the OD600 reached 0.6. Isopropyl-β-D-thiogalactoside
(IPTG) was then added at a final concentration of
0.4 mmol/L. The cells were further incubated at 28 °C
for approximately 6 h and harvested by centrifugation
(8,000 rpm, 5 min). The cell suspension was treated
twice with a French Pressure Cell (Thermo, USA) at
15,000 psi and centrifuged at 13,000 rpm for 10 min.
The extract was further purified using the “IMAC”
protein purification system following the manufacturer’s instructions (Invitrogen).
in the B. thuringiensis spore-ICP preparation was
quantified following standard procedures (National
Standard of the People’s Republic of China, GB/T
19567.2-2004)
using
sodium
dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and comparative densitometry of the 130-kDa ICPs
bands on SDS-PAGE gels from the prepared powder
and that of a standard reference spore-crystal powder
(with 8.0% ICPs, KeNuo Biotech Co. Ltd, Wuhan,
China).
B. thuringiensis spore-ICP preparation and
measurement of ICPs concentration
Characterization of the purified wild-type and
mutant chitinases
A seed culture of B. thuringiensis YBT-1520 (or
YBT-020) grown at 30 °C for 10 h was used as an inoculum. The fermentation of YBT-1520 (or YBT-020)
was carried out in a 15-L fermentor (with a 7.5-L LB
broth loading) by inoculating with 7.5 ml of seed
culture. The culture was kept at 30 °C and with a stirring rate of 400 r/min until ~ 70% spores and crystals
released from lytic cells (about 30 h to 32 h). The
spore-crystal suspension was harvested, and was then
subjected to rapid spray desiccation for preparation of
the wettable spore-crystal powder. The ICPs content
Analytical assays
The cell density at 600 nm was measured using a
UV-VIS spectrophotometer (DU-800 Nucleic Acids/Protein Analyzer, Beckman Coulter). Purified B.
thuringiensis wild-type and mutant chitinases were
quantified according to Bradford [25] using bovine
serum albumin as the standard. The expression of
wild-type or mutant chitinases in E. coli was analyzed
by SDS-PAGE in 10% polyacrylamide gels as described by Laemmli [26]. Prior to the chitinolytic activity assay, colloidal chitin was prepared from farinose chitin (TaKaRa) following the procedures described by Zhang et al. [27]. The chitinase activity of
the wild-type and mutant chitinases was assayed following a previously published method [28], except
the modified reaction mixture contained 200 μL of 2%
colloidal chitin (pH 6.8), 500 μL of diluted purified
Chi (pH 6.5), and Tris-HCl buffer (pH 7.4) to a final
volume of 1.0 mL (pH 6.8). The reaction mixture was
incubated at 37 °C for 1 h. After centrifugation, 500 μL
of the supernatant fluid was added to 1 mL of dinitrosalicylic acid (DNS) reagent [29] and incubated in
boiling water for 10 min. The absorbance of the mixtures was measured at 535 nm, and the reducing
sugars in the mixtures were calculated by comparison
to a calibration curve constructed using pure NAG
(Sigma). One unit of chitinase activity was defined as
the amount of enzyme that produced reducing sugars
equivalent to 1 μmol of NAG under the above conditions.
The effect of pH on purified chitinase activity
was determined in a series of citrate-phosphatebuffered reaction solutions containing 300 µL citrate-phosphate buffer, 200 µL colloidal chitin and 500
µL purified chitinase at pH values ranging from 2.0 to
9.0. The chitinolytic reaction was performed at 37 °C
for 1 h. The enzyme activities at various pH values
were determined using the assay described above.
The effect of temperature on the activity of purified chitinase was determined by incubating the reaction solutions at a range of temperatures (25 °C, 30 °C,
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Int. J. Biol. Sci. 2015, Vol. 11
35 °C, 37 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 70 °C,
and 80 °C) for 1 h. The chitinase activity was measured using the method described above.
The effect of metal ions, including Fe2+, Ca2+,
2+
Zn , Pb2+, Mg2+, Mn2+, and Cu2+, at final concentrations of 1 mmol L-1 on chitinase activity was determined by incubating the reaction solutions containing
each metal ion at 37 °C for 1 h. The enzyme activity
was then measured following the method described
above.
Toxicity bioassay
For toxicity bioassays against H. armigera, 1.00 g
B. thuringiensis YBT-1520 spore-ICP powder was
added to 10 mL 0.01 M sterile Na2CO3⋅NaHCO3 buffer
(pH 9.6), and stirred vigorously to allow complete
dissolution of the crystals, then diluted with sterile
double distilled water (ddH2O) to 25.0 mL using a 25
mL volumetric flask to prepare a 40.0 mg/mL
spore-ICP powder stock solution. The stock solution
was mixed with 0.01 M PBS (pH7.4)-dissolved purified chitinase (Chi9602 or ChiW50A, each at the concentration of 280 µg/mL) at a ratio of 7 : 1 (V/V) to
prepare the composite formulations containing
spore-ICP powder and chitinase at the final concentration of 35 mg/mL and 35 µg/mL, respectively.
YBT-1520 spore-ICP solution at the final concentration
of 35 mg/mL was prepared in parallel by adding 1
aliquot ddH2O to 7 aliquots of powder stock solution.
Bioassays of spore-ICP preparation and the composite
formulations against neonate larvae of H. armigera
were performed as previously described [30]. The 50%
lethal concentration (LC50) was calculated for each
treatment.
For bioassays against C. elegans, 1.00 g B. thuringiensis YBT-020 spore-ICP powder was dissolved with
10 mL sterile 0.01 M Na2CO3⋅NaHCO3 buffer (pH 9.6),
and diluted with sterile ddH2O to 25.0 mL using a 25
mL volumetric flask to prepare a 40.0 mg/mL
spore-ICP powder stock solution. The stock solution
was further diluted to 2.0 mg/mL, 5.0 mg/mL, 20.0
mg/mL and 30.0 mg/mL solutions with sterile
ddH2O. Each 100 µL of above diluted solutions (including the stock solution) was added to different
wells of a 96-well cell culture plate, which were
loaded with 50 µL 80 µg/mL chitinase (Chi9602 or
ChiW50A), 5 µL C. elegans larvae (L4) suspension
(containing a total of 40 larvae), 5 µL 10 mM
5-fluoro-2'-deoxy-uridine, and 40 µL E. coli OP50 cell
suspension (at OD600 of 0.6). In parallel, each 100 µL of
above solutions was added to wells of another plate
loading with similar solutions except 50 µL chitinase
was substituted with 50 µL sterile ddH2O. The bioassays against C. elegans were performed according to
308
the method described by Dengg and van Mell [31],
except that synchronized L4 larvae were used. Lethality was evaluated after three days by probing
larvae with a dissecting needle under a stereo microscope. Percentage of lethality was calculated for each
treatment. Purified chitinases Chi9602 and ChiW50A
at the concentrations of 0−800 µg/mL were used as
the controls.
Antifungal assays
Target fungal strains were inoculated onto the
central spot of each PDA Petri plate and cultivated for
24-48 h at 28 °C. A sterile filter paper (diameter, 8 mm)
spotted with 50 µL of the purified chitinases at the
final concentration of 200 µg/mL (filtered with a 0.45
µm sterile filter membrane) or 50 µL sterile H2O was
placed on the surface of each plate. The plates were
then incubated at 28 °C until inhibition of the hyphal
extension around the filter papers was observed. The
diameters of each inhibition zone were then measured
for activity evaluation.
RESULTS
Chi9602 homology modeling
Most available B. thuringiensis genomes contain
chitinase-encoding sequences. Based on the sequence
of the B. thuringiensis BMB171 chitinase gene [32], a
chitinase-encoding gene was amplified from the genome of B. thuringiensis subsp. tenebrionis YBT-9602 by
PCR. Nucleotide sequence analysis and nucleotide
alignment using the online BLASTN tool suggested
that this 2031-bp gene, designated chi (GenBank accession no. KF671757.1), is a chitinase-encoding
member of the family 18 glycosyl hydrolases. The chi
gene encodes a 676-amino acids protein (Chi9602)
with a deduced molecular mass of 74.5 kDa and an
isoelectric point of 5.89. Conserved domain architecture analysis of Chi9602 revealed that the protein has
a modular structure composed of an N-terminal chitin-catalytic domain (CCD, from amino acids 42 to
452), a fibronectin type-III-like domain (Fn3D, from
amino acids 504 to 558), and a C-terminal chitin-binding domain (CBD, from amino acids 582 to
646) (Fig. 1A). The first 34 residues of the N-terminus
were predicted to be a signal sequence by SignalP,
with a possible cleavage site located between amino
acids 34 and 35. Sequence alignments of chi with the
chitinase gene of subsp. kurstaki BMB171 (GenBank
accession no: CP001903.1), chiA of subsp. kurstaki
HD-73 (GenBank accession no: EF581163.2), and chiA
of subsp. colmeri 15A3 (GenBank accession no:
DQ512474.1) revealed sequence identity in excess of
97%.
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Int. J. Biol. Sci. 2015, Vol. 11
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Figure 1. Schematic illustration of Chi960235-459 homology modeling and Chi960235-459 and (NAG)2 molecular docking. A, Structural organization of
chitinase Chi9602. Abbreviations: AA, amino acid; SS, signal sequence; CCD, chitin-catalytic domain; Fn3D, fibronectin type-III-like domain; CBD, chitin-binding
domain. B, Orthogonal view of the Chi960235-459 ribbon model. The colored α-helixes (red) and β-sheet (yellow) constituting the (βα)8 TIM barrel structure are
shown. C, Surface view of the spatial structure of Chi960235-459. The substrate-binding semi-closed groove channel is indicated by the rectangular frame. D, Molecular
structure of the N-acetyl-D-glucosamine (NAG) dimer. E, The docking model of the Chi960235-459-(NAG)2 complex. The bound red/blue-colored spheroid molecule
of (NAG)2 shows the position of the substrate binding groove. F, The docking model of the Chi960235-459-(NAG)2 complex, showing the residues selected for
site-directed mutagenesis.
To identify a reference protein for homology
modeling of Chi960235-459, the PDB database was
searched for homologous proteins. Among the suggested homologous proteins, ten proteins (Supplementary Material: Table S2) with 30% to 70% sequence similarity to Chi960235-459 were considered as
possible reference proteins. The sequences of these
proteins were aligned with the sequence of
Chi960235-459 (Supplementary Material: Fig. S2), which
revealed that Chi960235-459 harbored most of the conserved residues of the family 18 chitinases, including
the specific chitinase-binding motif “SXGG” and
conserved CCD motif “DXDXE”, which are essential
for function [23, 33-35]. One of these proteins, Bacillus
circulans chitinase A1 (PDB ID: 1ITX), had the highest
sequence identity (61%) to Chi960235-459, shared the
specific chitinase-binding motif “SVGG” and the
conserved CCD motif “DLDWE” with Chi960235-459,
and had a structure with a high resolution of 1.10 Å.
Thus, 1ITX was selected as the reference protein for
Chi960235-459
homology
modeling.
A
three-dimensional structural model of Chi960235-459
was constructed, and the structural coordinates of the
optimized Chi960235-457 model were evaluated by
Ramachandran plot [36] (Supplementary Material:
Fig. S3), which revealed that 93.4% residues (426 of
456 amino acids) were in favored regions and 97.6%
residues (445 of 456 amino acids) were in allowed
regions, indicating the favorability of this model. A
three-dimensional ribbon model of Chi960235-459 was
then plotted using PyMol (Fig. 1B), which revealed
the classical TIM-barrel structure of the chitinase CCD
consisting of 8 α-helices (α1 to α8, Fig. 1B) and 8
β-strands (β1 to β8, Fig. 1B) along with an α-helix-rich
terminal domain and an α+β domain, as has been
observed in other family 18 chitinases [23, 37, 38]. The
barrel fold composed of β-strands formed a long surface groove, with α-helices and coils connecting the
strands. A surface view of the Chi960235-459 spatial
model (Fig. 1C) clearly showed a channel of this
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Int. J. Biol. Sci. 2015, Vol. 11
groove that is predicted to be involved in substrate
binding and a “bed” structure formed by the base of
the TIM barrel, consistent with the structure of Yersinia entomophaga Chi1 [23].
The β4 strand of the TIM-barrel contained the
“DLDWE” motif, a characteristic sequence that includes a conserved Glu residue with a crucial role in
enzyme function in several other chitinase family 18
members because this Glu residue protonates the
oxygen in the scissile glycosidic bond [34, 38-40].
Thus, we refer to this residue, E211 in Chi9602, as a
key residue for targeting the orientation range of the
residues that interacts with the substrate and modulates enzyme activity during molecular docking.
Molecular docking of Chi9602
Family 18 chitinases hydrolyze C2 N-acetylated
β(1-4)-linked substrates, and catalysis occurs until the
substrate is hydrolyzed [38, 40]; therefore, we mimicked a dimer fold of NAG (Fig. 1D) to use as the
substrate for molecular docking. Flexible docking of
Chi960235-459 toward the substrate (NAG)2 was performed to obtain a matched conformation of the
Chi960235-459 + (NAG)2 ligand complex (Fig. 1E; Supplementary Material: Fig. S4A). As expected, (NAG)2
matched well to the Chi960235-459 homology model by
fully implanting into the groove fold (Fig. 1E). While
E211 was set as the core site for activity, a docking
area around E211 comprising 27 amino acid residues
clearly revealed the coverage of the full groove fold
embedding the ligand (NAG)2 (Supplementary Material: Fig. S4A, S4B). To select the candidate residues
for mutagenesis to enhance chitinase activity, we
considered hydrophilic or hydrophobic residues that
could form hydrogen bonds or hydrophobic bonds
with (NAG)2, and residues that were oriented toward
or surrounding the (NAG)2 with relatively greater
steric hindrance. Ten residues, Y46, W50, R55, W171,
E211, D287, D385, R343, W447, and S450, predicted to
be involved in forming the substrate-binding groove
structure and packing the substrate were selected as
target residues for site-directed mutagenesis.
310
of the mutated codons (Y46A, W50A, R55A, W171A,
E211A, D287A, R343A, D385A, W447A, and S450A)
were obtained by substituting each target amino acid
codon with a codon encoding Ala. SDS-PAGE analysis demonstrated that wild-type Chi9602 and the
mutant Chi9602 variants were expressed as a monomeric protein component with the predicted size (~ 70
kDa) in the cytosol (Supplementary Material: Fig.
S5A, S5B, and S5C). These chitinases were then purified from each transformed E. coli strain.
The chitinase activities of purified wild-type and
mutant Chi9602 variants were measured under normalized conditions using colloidal chitin as the substrate. As shown in Fig. 2, the mutants ChiR55A and
ChiD385A showed almost the same level of activities
with Chi9602, and most of other mutants exhibited
decreases in enzymatic activity ranging from about
10% to about 90% compared to Chi9602 (51.8 U/mg);
however, three mutant enzymes exhibited increased
activity: ChiW50A, which contains a mutation in the
substrate-binding domain, and ChiD385A and
ChiS450, which contain mutations in the catalytic
domain, exhibited increases in activity of 62%, 15%
and 51%, respectively.
Given the substantially increased activity of
ChiW50A and ChiS450A, we constructed the double
mutant ChiW50A/S450A. However, this double mutant enzyme did not exhibit synergistically increased
activity but rather a decrease in activity of approximately 19%, 50%, and 47% compared to Chi9602,
ChiW50A, and ChiS450A, respectively.
Site-directed mutagenesis and activity of mutant chitinases
Site-directed mutagenesis was conducted to
mutate all selected target residues to Ala. Ala was
chosen because it is an electrically uncharged hydrophobic amino acid with less steric hindrance. Ala is
likely appropriate for probing residues that modulate
enzyme activity through spatial effects rather than by
forming hydrogen bonds or hydrophobic bonds,
thereby facilitating the identification of potential activity-enhancing mutations. Using the PCR-SOE
strategy, ten mutated Chi9602 variants containing one
Figure 2. Measurement of the specific enzyme activity of the mutant chitinases
generated by site-directed mutagenesis.
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Int. J. Biol. Sci. 2015, Vol. 11
Effect of pH, temperature and metal ions on
ChiW50A enzyme activity
Due to its relatively higher activity, the
ChiW50A mutant was selected for further studies to
determine whether the mutation altered the pH,
temperature and metal ion requirements compared to
wild-type Chi9602. ChiW50A retained relatively
higher activity at pH 5 to 8, with highest activity at pH
7, consistent with Chi9602 (Fig. 3A). Interestingly, as
shown in Fig. 3B, ChiW50A exhibited higher activity
across the temperature range of 25-37 °C compared to
Chi9602; however, ChiW50A activity rapidly decreased across 40 °C to 60 °C and was inactivated at
temperatures above 60 °C. As the temperature increased from 25 °C to 55 °C, ChiW50A lost approximately 95% of its activity. Chi9602 exhibited greater
thermostability and lost only 10% of its activity at
similar temperatures, appearing relatively stable
across a temperature range of 25-55 °C. Fig. 3C shows
that Cu2+ and Zn2+ had remarkable inhibitory effects
on ChiW50A and Chi9602 activity, whereas Mg2+ and
Ca2+ increased enzymatic activity by approximately
15% to 20%. Limited effects of Fe2+ and Mn2+ were
observed, but Pb2+ appeared to inhibit the activity of
ChiW50A more strongly than Chi9602 activity.
311
sults demonstrate that the ChiW50A mutant has
greater synergistic lepidopteran-larvicidal and nematicidal activities compared to wild-type Chi9602.
Synergistic lepidopteran-larvicidal and nematicidal activities of ChiW50A
One of most promising applications of chitinases
in biocontrol is to enhance the pest-larvicidal activity
of B. thuringiensis toxins [8]. To determine if the
ChiW50A mutant enhanced lepidopteran-larvicidal
activity when added to insecticidal spore-ICP preparations of a lepidopteran-active B. thuringiensis
YBT-1520 strain, we performed normalized bioassays
with mixtures of the purified chitinases and YBT-1520
spore-ICP preparations against H. armigera larvae.
Neither wild-type Chi9602 nor ChiW50A had larvicidal activity; however, both Chi9602 and ChiW50A
exhibited synergistically increased activities when
added to the YBT-1520 preparations, resulting in decreases in the LC50 of 17.6% and 30.4%, respectively
(Table 1). The synergistic nematicidal activities of
wild-type Chi9602 and the mutated ChiW50A against
C. elegans larvae when added to preparations of a
nematicidal B. thuringiensis YBT-020 strain were also
evaluated. As shown in Fig. 4, both Chi9602 and
ChiW50A mutant possessed no nematicidal activity
alone; however, when added to the B. thuringiensis
YBT-020 spore-ICP preparation, ChiW50A exhibited a
steadily increasing pattern of greater synergistic activity compared to Chi9602 at the YBT-020 concentration range of 1−14 mg/mL. Overall, 100% mortality
was observed when both enzymes were added to 15
mg/mL YBT-020 spore-ICP preparations. These re-
Figure 3. Effects of pH, temperature and metal ions on the chitinase
activity of ChiW50A and wild-type Chi9602.
Antifungal assays
The antifungal activities of purified ChiW50A
and Chi9602 were investigated. Both enzymes exhibited obvious inhibitory effects on hyphal growth by
the target fungi as visualized by their obvious inhibition zones compared to negative controls (Fig. 5). By
comparing the diameters of inhibition zones,
ChiW50A showed relatively greater activities against
Sclerotinia sclerotiorum, Fusarium oxysporum, Fulvia
fulva, and Botrytis cinerea compared to Chi9602, but the
difference in activity against Physalospora piricola and
Rhizoctonia solani between the two enzyme variants
was not remarkable.
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Int. J. Biol. Sci. 2015, Vol. 11
312
Table 1. Bioassays of the insecticidal mixtures of purified chitinases and YBT-1520 spore-ICP preparations against Helicoverpa armigera.
Samples a
Linear regression equation b
Linearly dependent coefficient b
Chi9602
ChiW50A
YBT-1520
YBT-1520 + Chi9602
YBT-1520 + ChiW50A
−
−
Y= -5.207+2.064X
Y= -8.315+3.192X
Y= -5.963+2.460X
−
−
0.9444
0.9653
0.9995
LC50
(μg·mL-1)
>1000
>1000
4.70
3.88
3.27
95% confidence bounds
(μg·mL-1)
−
−
4.058~5.466
3.384~4.413
2.826~3.719
Note: a, Sample initial concentration: Chi9602, 35 µg/mL; ChiW50A, 35 µg/mL; YBT-1520, 35 mg/mL; YBT-1520 + Chi9602/ChiW50A, 35 mg/mL (YBT-1520) and 35 µg/mL
(Chi9602 or ChiW50A) at the final concentration. The sample solutions were diluted to 1, 1/2, 1/4, 1/8 and 1/16 diluents with sterile ddH2O for bioassays; b, PROBIT model of lethal-concentration.
Figure 4. Synergistic effect of the ChiW50A mutant on the nematicidal activity of B. thuringiensis YBT-020 spore-ICP preparations against C.
elegans larvae. B. thuringiensis YBT-020 spore-ICP powder was prepared as 1.0 mg/mL, 2.5 mg/mL, 10.0 mg/mL, 15.0 mg/mL and 20.0 mg/mL solutions, respectively.
Each 20 μg/mL purified ChiW50A or Chi9602 at the final concentration was added in above solution to prepare the composite formulations.
Figure 5. Antifungal activity of the ChiW50A mutant and the Wild-Type Chi9602. A, Sclerotinia sclerotiorum FB014; B, Physalospora piricola FB016; C,
Fusarium oxysporum FB012; D, Fulvia fulva FB009; E, Botrytis cinerea FB007; F, Rhizoctonia solani FB010. Labels 1, 2 and 3 in each plate indicate the treatments using sterile
H2O (the negative control), ChiW50A and Chi9602 (at the final concentrations of 200 µg/mL), respectively.
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Int. J. Biol. Sci. 2015, Vol. 11
DISCUSSION
Chitin is a major structural component in various
insect tissues, including the parietal layer, exo- and
endocuticle, respiratory tract, peritrophic membrane
and intestinal tract. Chitin functions as a mechanically
strong supporting scaffold material as well as a permeability barrier lining the trachea and gut epithelium [41]. Chitin is also an integral part of the nematode
eggshell and pharyngeal lumen walls [42] and a major
cell wall component of many plant-pathogenic fungi
[43]. Bacterial chitinases can hydrolyze various chitin-based substrates [10, 11, 44] and exhibit synergistic
activity with B. thuringiensis spore-ICP preparations
against pest larvae [9, 10]. The biological properties of
bacterial chitinases, particularly the chitinase produced by insecticidal B. thuringiensis, indicate their
potential for biocontrol of agriculturally important
lepidopteran and nematode pests as well as
plant-pathogenic fungi.
The molecular docking model permits an intuitive explanation of the binding of the enzyme and
ligand molecules, suggesting candidate sites for
site-directed mutagenesis [45, 46]. To identify potential active-site residues in chitinases, many previous
site-directed mutagenesis studies have substituted
substrate-binding residues with hydrophilic or hydrophobic residues that form hydrogen bonds or hydrophobic bonds with the substrate, frequently resulting in decreased enzymatic activity [41-43]. In this
study, when E211 was fixed as the central site of the
Chi960235-459-(NAG)2 complex model, 27 amino acid
residues were identified as the pocket residues forming the substrate-binding groove structure by the
Discovery Studio Visualizer tool (Supplementary
Material: Fig. S4B). Due to its lack of electric charge
and reduced steric hindrance, we chose the uncharged
amino acid Ala as the replacement codon in the
site-directed mutagenesis experiments. Moreover, we
considered both the pocket residues and the substrate-binding residues as the target codons. Based on
their hydrophilic/hydrophobic nature as well as their
spatial positions and steric hindrance, we selected 10
residues, including substrate-binding residues (e.g.,
E211) and residues surrounding the substrate-binding
residues (e.g., W50). Our results demonstrated that
although most of the mutations (Y46A, R55A, W171A,
E211A, D287A, R343A, and W447A) decreased activity, as expected, several of the mutations (W50A,
D385A, and S450A) increased chitinase activity. Interestingly, all three activity-enhancing mutations
involve residues that are relatively far from the substrate-binding fold, indicating that the initial selection
of these residues rather than residues close to the
center of the fold is reasonable.
313
Based on their conservation and effects on enzyme activity, these residues can be divided into three
groups. Residues in the first group, which include
Y46, W171, E211, D287, R343 and W447, are conserved, and their mutagenesis leads to remarkably
altered activity. Site-directed mutagenesis of these
residues consistently resulted in decreased enzyme
activity (Fig. 2). We proposed that these residues are
important for interactions with the substrate by
forming critical stabilizing interactions or are proximal to the enzyme-ligand binding sites [37, 47]. The
Y46 and W447 residues are apparently located below
the substrate molecule in the enzyme-substrate pocket
fold; therefore, they are likely to support the substrate
and stabilize the enzyme-substrate reaction configuration. A similar role was proposed for W171, which
is located above the pocket structure and restricts the
substrate within the appropriate enzyme-substrate
configuration. As indicated above, E211 is a core residue for enzyme activity. The E211 to A211 mutations
eliminates the hydrolysis of β-1,4-glycosidic bonds
although the enzyme binds to the substrate [39]. D287
and R343 are proposed to be important for substrate
delivery into the enzyme core center by forming hydrogen bonds with the substrates. The D287A and
R343A mutations hinder the substrate from entering
the core center of Chi9602, leading to nearly complete
loss of enzyme activity.
The second group of residues includes R55 and
D385, which are non-conserved and flank the enzyme-substrate complex fold. As shown in Supplementary Material: Fig. S4B, R55 is apparently located
outside the pocket fold that is relatively far from the
substrate molecule; therefore, mutation of this residue
slightly decreased enzyme activity. However, the
mutation of D385 to A385 eliminated negative charge;
the residue was closer to the pocket fold, increasing
the compactness and stability of the enzyme-substrate
complex and thereby increasing enzymatic activity.
The third group of residues includes
non-conserved residues that are critical for enzymatic
activity, such as W50 and S450. W50 is located in the
bottom of the pocket structure, and mutation of W50
to A50 vacates lateral chain space by replacing the
larger imidazole group-containing tryptophan residue with the smaller methyl group-containing alanine. The W50A mutation likely enables deeper insertion of the substrate into the groove fold closer to the
catalytic center, thereby possibly increasing the affinity between the substrate and enzyme and increasing
enzyme activity. The S450A mutation is located in the
outer area of the pocket structure and eliminates the
hydrophilicity associated with S450, thereby potentially shifting the positions of several adjacent residues (possibly E448 and W447) through the formation
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Int. J. Biol. Sci. 2015, Vol. 11
of hydrogen bonds. This hydrogen bond formation
causes the substrate to move closer to the catalytic
active center and increases enzymatic activity.
To determine if the ChiW50A mutant requires
similar conditions for enzymatic activity as wild-type
Chi9602, we compared the effects of pH, temperature
and metal ions on the activity of both enzymes. Our
results indicated that the W50A mutation does not
radically alter these requirements. This finding suggests that the strategy used in this study is a
well-controlled and straight-forward method to
modulate only the enzymatic activity of target enzymes without affecting other properties.
We further demonstrated that the ChiW50A chitinase mutant exhibited greater synergistic effects
than wild-type Chi9602 in controlling H. armigera larvae
in laboratory-based trials. Although various bacterial
chitinases exhibited synergistic effects on lepidopteran pests, the relatively low activity of naturally
occurring bacterial chitinases has hindered their application in agricultural practices. The results presented here suggest an approach to improve chitinase
activity that may be applicable to other lepidopteran-pest biocontrol processes. Moreover, the mutated
ChiW50A also exhibited an enhanced synergistic effect on the nematicidal activity of B. thuringiensis
spore-ICP preparations; such an effect on nematicidal
activity has not been reported previously and should
be validated for other nematodes, particularly
plant-parasitic nematodes such as the root-knot nematode Meloidogyne incognita, an economically important agricultural pest.
In conclusion, the current study reports a
goal-orientated approach to enhance the activity of a
B. thuringiensis chitinase through homology modeling,
molecular docking and site-directed mutagenesis.
One of the resulting mutant chitinases exhibited a
greater than 60 % increase in enzymatic activity
without radically altered pH, temperature and metal
ion requirements and exhibited synergistic effects on
the control of lepidopteran and nematode pests and
antifungal activity in laboratory trials. Given the simplicity and reliability of the methodology employed
here, this strategy is appropriate for the molecular
modification of other enzymes.
314
work was supported by grants from the National
Basic Research Program of China (973 Program, grant
no. 2013CB127504), the National Science Foundation
of China (Grant No. 31270158), the Natural Science
Foundation of Hubei Province, China (Grant No.
2012FFA055), and the Non-Profit Science and Technology Research Funds of Hubei Province of China
(Item no. 2012DBA10001). This work was also supported by the Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University (Grant
No. AMLKF201002).
COMPETING INTERESTS
The authors have declared that no competing
interest exists.
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