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7/28/2019 10b-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides
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Functional characterization, homology modeling and dockingstudies ofb-glucosidase responsible for bioactivation
of cyanogenic hydroxynitrile glucosidesfrom Leucaena leucocephala (subabul)
Noor M. Shaik Anurag Misra Somesh Singh
Amol B. Fatangare Suryanarayanarao Ramakumar
Shuban K. Rawal Bashir M. Khan
Received: 10 April 2012 / Accepted: 8 October 2012 Springer Science+Business Media Dordrecht 2012
Abstract Glycosyl hydrolase family 1 b-glucosidases are
important enzymes that serve many diverse functions inplants including defense, whereby hydrolyzing the defen-
sive compounds such as hydroxynitrile glucosides. A
hydroxynitrile glucoside cleaving b-glucosidase gene
(Llbglu1) was isolated from Leucaena leucocephala,
cloned into pET-28a (?) and expressed in E. coli BL21
(DE3) cells. The recombinant enzyme was purified by Ni
NTA affinity chromatography. The optimal temperature
and pH for this b-glucosidase were found to be 45 C and
4.8, respectively. The purified Llbglu1 enzyme hydrolyzed
the synthetic glycosides, pNPGlucoside (pNPGlc) and
pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed
amygdalin, a hydroxynitrile glycoside and a few of the
tested flavonoid and isoflavonoid glucosides. The kinetic
parameters Km and Vmax were found to be 38.59 lM and
0.8237 lM/mg/min for pNPGlc, whereas for pNPGal the
values were observed as 1845 lM and 0.1037 lM/mg/min.
In the present study, a three dimensional (3D) model of the
Llbglu1 was built by MODELLER software to find out the
substrate binding sites and the quality of the model was
examined using the program PROCHEK. Docking studies
indicated that conserved active site residues are Glu 199,
Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462.
Docking of rhodiocyanoside A with the modeled Llbglu1resulted in a binding with free energy change (DG) of
-5.52 kcal/mol on which basis rhodiocyanoside A could
be considered as a potential substrate.
Keywords Glycosyl hydrolase family 1 Molecular
docking Homology modeling Leucaena leucocephala
Abbreviations
GH1 Glycosyl hydrolase family 1
IPTG Isopropyl-b-D-thiogalactoside
pNPGlc p-Nitrophenyl-b-D-glucopyranoside
pNPGal p-Nitrophenyl-b-D-galactopyranoside
Introduction
Glycoside hydrolases are widely distributed enzymes that
hydrolyze the glycosidic bond between two or more carbo-
hydrates or between a carbohydrate and non-carbohydrate
moiety. Based on the sequence similarities, these enzymes
have been classified into115 families, whose unique features
and representative members are described in the Carbohy-
drate-Active enzymes database (http://www.cazy.org/) [1].
Plant b-glucosidases belonging to Glycosyl hydrolase fam-
ily 1 (GH 1), serve a number of diverse and important
functions, including bioactivation of defense compounds
[26], cell wall degradation in endosperm during germina-
tion [7], activation of phytohormones [8, 9] and lignifica-
tions [10, 11]. In addition,b-glucosidases also play a key role
in aroma formation in tea, wine, and fruit juices [1214].
Plants produce innumerable secondary metabolites involved
in defense against pathogens and herbivores. These defense
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-012-2179-6) contains supplementarymaterial, which is available to authorized users.
N. M. Shaik S. Singh A. B. Fatangare
S. K. Rawal B. M. Khan (&)
Plant Tissue Culture Division, National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune 411008, India
e-mail: [email protected]
A. Misra S. Ramakumar
Department of Physics, Bioinformatics Centre, Indian Institute
of Science, Bangalore 560012, India
123
Mol Biol Rep
DOI 10.1007/s11033-012-2179-6
http://www.cazy.org/http://dx.doi.org/10.1007/s11033-012-2179-6http://dx.doi.org/10.1007/s11033-012-2179-6http://www.cazy.org/7/28/2019 10b-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides
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compounds are often stored as b-glucosides and bio-acti-
vated by specific b-glucosidases [2].
In higher plants, glycosylation serves to protect the plant
against the toxic effects of its own chemical defense system
wherein b-glucosidase confers resistance to pathogens and
herbivores by catalyzing the cleavage of these defensive
glucosides [15]. These b-glucosidases and glucosides are
considered to exist in different cellular compartments [16].Whenever the tissue undergoes an injury by a mechanical
damage or by infection, these enzymes come into contact
with the defensive glucosides and then by implicating their
hydrolytic action to release toxic aglycones such as
hydrogen cyanide, saponins, coumarins and naphthoqui-
nones for the execution of defense mechanism [17]. The
well characterized two-component defense systems include
a-hydroxynitrile glycosides (cyanogenic glycosides) in
different plants [6], benzoxazinoid glycosides in graminae
[18], avenacosides in Avena sativa [5], isoflavonoid gly-
cosides in legumes [17] and glucosinolates mainly in
brassicales [3]. The physiological functions of b-glucosi-dases varies greatly depending upon their origin (plants,
fungi, animals or bacteria) and substrate specificity.
A distinguished feature of the human cytostolic b-glu-
cosidase (hCBG) is its ability to hydrolyze many common
dietary xenobiotics, including glycosides of phytoestro-
gens, flavonoids, simple phenolics and cyanogens [19, 20].
The hCBG shows high specificity for 40- and 7-glucosides
of isoflavones, flavonols, flavones and flavonones, but
does not hydrolyze 3-linked flavonoid glucosides [20]. A
b-glucosidase from soybean and okara shows the speci-
ficity towards glucosyl isoflavones [21].
Among the plants harboring cyanogenic glycosides,
several of them produce b- and c-hydroxynitriles also.
Because of the striking structural similarities amonga-, b-,
and c-hydroxynitriles and a high frequency of co-occur-
rence, it has been proposed that the compounds are bio-
synthetically related to each other [22, 23]. These
hydroxynitrile glycosides are bioactivated by specific
b-glucosidases. The hydroxynitrile cleavingb-glucosidases
have been well characterized from wide variety of plants
such as Trifolium [24], Cassava [25], Prunus [26], Viciacin
[27] and Lotus sp [28].
b-glucosidases having different three dimensional struc-
tures, share the overall fold of the catalytic domain in GH
super-family. The families GH 1, GH 5, and GH 30 belongs
to the clan GH-A, and they all have similar (b/a)8 barrel
domains that contain their active site residues [29, 30]. The
length and subunit masses of these GH 1 enzymes vary
considerably, depending upon the presence of domains and
redundant GH 1 domains (as in human LPH), but the cata-
lytic domain itself ranges from around 440550 residues,
depending upon the lengths of the variable loops at the
C-terminal ends of the b-strands of the (b/a)8 barrel [30].
The GH 1 enzymes may have ratherbroad rangeof glycone
specificity, however, one enzyme may hydrolyze b-D-gluco-
sides, b-D-galactosides, b-D-fucosides, b-D-mannosides and
a-L-arabinosides, or may be specific for one or a few glycone
sugars. The basis of the tremendous diversity in function of
b-glucosidases, especially in plants, is the substrate aglycone
specificity differences that determine their natural substrate.
Structures of complexes of enzymes with inhibitors, andmutant enzymes with substrates, along with mutagenesis and
chimera studies comparing similar enzymes with divergent
specificities, have suggested that the basis of aglycone spec-
ificity is complex. Although this includes mutagenesis and
structural studies of human cytoplasmic b-glucosidase [31,
32]. The plant GH 1 enzymes have served as the primary
model, due to their high diversity in aglycone specificity.
Maize ZmGlu1 and Sorghum dhurrinase 1 (SbDhr1) are
closely related, displaying 70 % amino acid sequence iden-
tity, but have distinct specificities. ZmGlu1 has broad range
specificity, but cannot hydrolyze dhurrin,the natural substrate
of SbDhr1, while SbDhr1 hydrolyzes only dhurrin. Studies ofreciprocal ZmGlu1/SbDhr1 chimeric enzymes [33] and sub-
sequent structural and site-directed mutagenesis studies [34
37] indicated that aglycone specificity determining sites are
different in ZmGlu1 and SbDhr1.
Due to its unique genetic simplicity, the cyanogenic gly-
coside pathway has a pioneering status in the metabolic
engineering [38]. Engineering of the secondary metabolism of
plant defensive compounds is emerging as a novel approach
for the development of transgenic plants with the resistance
against insects and pathogens [39, 40]. Further, the avail-
ability of the genes encoding the biosynthetic enzymes of
secondary metabolism has made the transfer of entire bio-
synthetic pathways between plants feasible [41]. Therefore,
identification and characterization of genes involved in bio-
synthesis and bio-activation of hydroxynitrile compounds not
only define structure and function of theenzymes, but also can
find application in the genetic engineering of the crop plants
with resistance to insects and pathogens. In this paper, we
illustrate cloning, hetrologous expression, biochemical and
functional characterization ofLlbglu1 gene encoding a GH 1
b-glucosidase from L. leucocephala, a leguminous tree used
as a raw material for pulp and paper industry in India [42]. In
addition, to find the probable natural substrate for of the
Llbglu1, phylogenetic analysis, homology modeling and in
silico substrate docking studies were also performed.
Materials and methods
Plant material, micro organisms, vectors and enzymes
L. leucocephala K636 seeds were obtained from Indian
Tobacco Centre (ITC), Rajamundry, India. E. coli XL1
Mol Biol Rep
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strain (Stratagene, USA) was used as host for genetic
transformation. E. coli BL21 (DE3) (Novagen, USA) was
used as a host for heterologous expression of protein.
TRIZOL reagent (Invitrogen, USA) was used for isolation
of total RNA. For cDNA synthesis, BD Powerscript
Reverse Transcriptase (Clonetech Lab. Inc. USA) was
used. Taq polymerase for PCR amplification was pur-
chased from Sigma USA. All restriction enzymes and T4DNA ligase were used from Promega, USA. pGEM-T Easy
vector (Promega, USA) used for cloning of PCR amplified
products and pET-28a(?) (Novagen, USA) for expression
of protein. NiNTA agarose affinity column (Qiagen,
USA) was used for protein purification. All the substrates
used for enzyme assay were obtained from Sigma, USA,
unless otherwise specified.
Cloning of full length cDNA of GH1 b-glucosidase
Total RNA was isolated from one-week old in vitro grown
seedlings ofL. leucocephala using the TRIZOLreagent andthe first strand cDNA was synthesized by using BD pow-
erscript reverse transcriptase. PCR amplification was per-
formed with specific primers designed from Llbglu1
sequence having restriction sites for KpnI at forward pri-
mer and XhoI at reverse primer to facilitate cloning of the
nucleotide sequence of the L. leucocephala cDNA,
deposited in GenBank nucleotide sequence data base
(Accession No. EU328158). Specific primer sequences
used for Llbglu1 were 50-GGTACCATGATGAAGAAGG
TGATGGTAGTA-30 (sense) and 50-CTCGAGTTAATAT
TTTTGAAGGAAGTTCCTG-3 0 (antisense), where the
underlined sequences are the restriction sites for KpnI and
XhoI, respectively. PCR amplification was performed with
AccuTaq-LA DNA polymerase (Sigma, USA) under the
following conditions: 95 C for 5 min followed by, 35
cycles of, denaturation for 30 s at 95 C, an annealing for
30 s at 58 C with extension of 1.5 min at 72 C and the
sample was further incubated at 72 C for another 10 min.
The PCR product was purified with Gen EluteTM gel
extraction kit (Sigma, USA) and subcloned into pGEM-T
Easy vector (Promega, USA) and confirmed by DNA
sequencing.
Construction of phylogenetic tree of plant GH1
b-glucosidases involved in defense
For phylogenetic analysis, translated protein sequence of
Llbglu1 was used to construct a phylogenetic tree with all
known plant GH1 b-glucosidases, deposited in GenBank
database. All the 27 protein sequences present in a cluster,
containing Llbglu1 sequence, were taken separately and
Neighbor-Joining, rooted phylogenetic tree was con-
structed using Mega 4.0 [43] with 1000 bootstrap trials.
To study sequence similarities of Llbglu1 with 12 dif-
ferent hydroxynitrile cleaving b-glucosidases present in
three different clusters in the phylogenetic tree, a multiple
sequence alignment (MSA) was done with the Llbglu1
(GenBank Accession No. ABY48758) using program
ClustalW (http://www.ebi.ac.uk/Tools/clustalw2) and the
colored alignment figure was generated using ESPript 2.2
server (http://espript.ibcp.fr/ESPript). The protein sequen-ces used for MSA are LjBGLU2, Lotus japonicus b-glu-
cosidase D2 (GenBank Accession No. ACD65510);
LjBGLU4, Lotus japonicus b-glucosidase D4 (GenBank
Accession No. ACD65509); LjBGLU7, Lotus japonicus
b-glucosidase D7 (GenBank Accession No. ACD65511);
TrCBG, Trifolium repens linamarase (GenBank Accession
No. CAA40057); PsAH1precursor, Prunus serotina
amygdalin hydrolase isoform AH I (GenBank Accession
No. AAA93234); PsPH5, Prunus serotina prunasin
hydrolase isoform PH C precursor (GenBank Accession
No. AAL35324); PsPH1, Prunus serotina prunasin
hydrolase isoform PH I (GenBank Accession No.AAA93032); PsPH4, Prunus serotina prunasin hydrolase
isoform PH B precursor (GenBank Accession No.
AAL39079); HbLinamarase, Hevea brasiliensis b gluco-
sidase (GenBank Accession No. ABL01537); MeLina-
marase, Manihot esculenta linamarase (GenBank
Accession No. AAB22162); SbDhr1, Sorghum bicolor
dhurrinase (GenBank Accession No. AAC49177); SbDhr2,
Sorghum bicolor dhurrinase-2 (GenBank Accession No.
AAK49119).
Expression of Llbglu1 in E. coli and purification
The b-glucosidase found to have a signal peptide of 21
amino acids using program Signal P 3.0 (http://www.
cbs.dtu.dk/services/SignalP). The mature sequence of the
b-glucosidase without signal sequence was amplified with
AccuTaq-LA DNA polymerase by using primers contain-
ing EcoRI at forward primer 50-GAATTCGATGCAAC
AAATGATATTTCC-30 and NotI at reverse primer 50-
GCGGCCGCTTAATATTTTTGAAGGAAGTTCCTG-3 0.
The resulting PCR product was cloned into pGEM-T vector
and further it was sequentially cloned into EcoRI/NotI sites
of His6 tagged gene fusion vector pET-28a (?) (Novagen,
USA). The resulting plasmid construct was transformed
into E. coli, BL21 (DE3).
For the protein expression, a single transformed colony
was inoculated into 50 ml LB medium containing
50 lg/ml kanamycin. It was grown at 37 C until A600reached to 0.50.6. Protein expression was induced with
0.05 mM isopropyl-b-D-1-thiogalactopyranoside (IPTG)
while incubating for 9 h at 20 C. Resulting cells were
harvested by centrifugation and resuspended in lysis buffer
(10 mM Tris, pH 8.0, 150 mM NaCl). Cells were disrupted
Mol Biol Rep
123
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with an ultrasonic cell disruptor and suspension was
incubated with 0.1 mg/ml of lysozyme and centrifuged at
12,000 rpm for 10 min and the supernatant was analyzed
for b-glucosidase activity.
The soluble protein was used for purification with Ni
NTA agarose affinity column (Qiagen, USA). The binding
ofb-glucosidase to the NiNTA agarose beads was carried
out at pH 8.0 in 10 mM Tris buffer and washing of non-specific proteins at pH 6.3 in 50 mM citratephosphate
buffer and elution of the enzyme at pH 4.5 in 50 mM cit-
ratephosphate buffers. The purified fractions were ana-
lyzed on SDS-PAGE and the activity was monitored using
pNPGlc as a substrate.
Enzyme assay and analysis of the reaction products
The enzyme activity was assayed spectrophotometrically
using pNPGlc and other substrates (Table 1). Appropri-
ately diluted enzyme was incubated with substrate (1 mM
pNPGlc) in 50 mM citratephosphate buffer (pH 4.8) in afinal volume of 500 ll. The reaction was terminated by the
addition of 500 ll of 1.0 M Na2CO3. The p-nitrophenol
liberated was read as phenolate anion at 420 nm. The
concentration of p-nitrophenol was determined using a
molar absorption co-efficient of 1.77x104. One unit of the
enzyme is defined as the amount of enzyme that liberates
1 lmol of p-nitrophenol/min under the assay conditions.
For the flavonoid glycosides the reaction mixture con-
tained 20 lg of purified Llbglu1, 100 mM TrisHCl (pH 4.8)
and 70 lM substrates. The flavonoid glycosides were pur-
chased from Chromadex (www.chromadex.com) and
standards were purchased from Sigma Aldrich (Sigma, USA).
The reaction mixture was incubated at 45 C for 1 h and the
reaction mixtures of flavonoid glycosides were terminated
and extracted twice by the addition of equal volume of ethyl
acetate.The ethyl acetate was then evaporated to dryness.The
dried reaction product was dissolved in methanol. The reac-
tion product was analyzed by high performance liquid chro-
matography (HPLC, Perkin Elmer, USA) equipped with adiode array detector (DAD) and a Waters symmetry C18
column (5 lm particle size, 4.6 mm 9 25 cm, supelco ana-
lytical, Sigma, USA). For generation of an analytical scale,
the mobile phase was consisted of sterile milliQ waterand was
programmed as follows 10 % acetonitrile for 5.0 min; 30 %
acetonitrile for 5.0 min; 60 % acetonitrile for 5.0 min and
90 % acetonitrile for 5.0 min. The flow rate was kept as 1 ml/
min and UV detection was performed at 260340 nm [44].
For amygdalin LCMS data was recorded on UPLC coupled
mass spectrometer (Waters, USA).
Recombinant enzyme characterization
Estimation of the recombinant b-glucosidase activities at
different pH and temperatures were conducted using the
purified enzyme. To determine the optimal pH, different
buffers in pH range of 3.57.0 were used. The buffers used
were 50 mM citratephosphate buffer (pH 3.56.0) and
100 mM phosphate buffer (pH 6.57.0). The b-glucosidase
activity was determined at standard assay conditions. Tem-
perature optimum was determined by measuring the activity
of the enzyme in 50 mM citratephosphate buffer (pH 4.8)
for 20 min at temperature ranging from 30 to 55 C with
5 C increments. To estimate pH stability, the enzyme was
pre-incubated in different buffers with pH range 2.012.0 at
37 C for varied time intervals. The residual b-glucosidase
activity was determined at standard assay conditions.
Hydrolytic activities of the recombinant enzyme towards
different pNP sugars (Table 1) were performed in 50 mM
citratephosphate buffer under standard assay conditions
(pH 4.8, 45 C). Kinetic parameters of the recombinant
enzyme Km and Vmax towards pNPGlc and pNPGal were
calculated from the MichaelisMenten equation.
Homology modeling and comparision of 3D model
with homologous structure
The 3D structure of Llbglu1 was built by homology mod-
eling based on high resolution crystal structure of homolo-
gous protein. To find the homologous structure in protein
data bank (PDB), the primary sequence of Leucaena
b-glucosidase was searched against PDB using BLASTP
program at NCBI (http://www.ncbi.nlm.nil.gov/blast).
Among all the homologs, cyanogenic b-glucosidase from
whiteclover(Trifolium repens, PDB: 1CBG) was found closest
Table 1 Activity of the purified recombinant b-glucosidase with
various nitro-phenyl derived chromogenic substrates
S.No Substrate Relative
activitya (%)
1 p-Nitrophenyl b-D-glucopyranoside 100
2 p-Nitrophenyl b-D-glucoronide 0.8
3 p-Nitrophenyl-N-acetyl-1-thio-b-
glucosaminide
0.5
4 p-Nitrophenyl a-D-glucopyranoside 0.1
5 p-Nitrophenyl N-acetyl-b-D-glucosaminide 4.0
6p
-Nitrophenylb
-D
-galactopyranoside 53.07 p-Nitrophenyl b-D-mannopyranoside 2.2
8 p-Nitrophenyl b-D-xylopyranoside 0.6
9 p-Nitrophenyl b-L-arabinopyranoside 6.0
10 o-Nitrophenyl b-D-glucopyranoside 42.0
11 p-Nitrophenyl N-acetyl-a-D-glucosaminide 0.4
a The purified b-glucosidase was incubated at optimum pH (4.8) with
potential substrates provided at 5 mM final concentration. Enzyme
activity was determined by measuring the rate of pNP (or oNP)
production spectrophotometrically at 420 nm. Reaction rates are
expressed here as a percentage of that observed with pNPGlc
Mol Biol Rep
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to Llbglu1, thus the three dimensional coordinates of white
clover b-glucosidase structure (1CBG) were used as a template
to generate a 3D model of the Llbglu1 using the program
MODELLER [45]. Modeled 3D structure was visualized with
program PyMoL [46] and quality of the model was examined
using the program PROCHEK [47]. In order to compare sec-
ondary structural elements (a-helices, b-sheets and turns) of
Llbglu1 with that of 1CBG, thepair wise sequencealignment ofthese two along with the model of Llbglu1 were used as input
for web based program ESPRIPT [48].
Pairwise structural alignment of modeled Leucaena
b-glucosidase was done with Trifolium 1CBG using com-
binatorial extension algorithm at SDSC-CE (http://www.cl.
sdsc.edu/ce.html) [49]. CE-MC (http://pathway.rit.albany.
albany.edu/*cemc) multiple protein structure alignment
server provides a web based facility for the alignment of
multiple protein structures (known/modeled PDBs) based on
Ca-coordinate distances using combinatorial extensions
(CE) and monte carlo optimization methods [50, 51]. 10
structures of family 1 b-glucosidase (most of them are ofplant origin) were aligned with that of modeled Llbglu1 to
compare the important residues involved in glycone binding
and catalysis.
Substrate binding studies through molecular docking
Molecular docking calculations were performed for the
modeled enzyme with pNPGs and a few natural substrates
(reported for closely related glucosidases) by using the DS
Modeling 1.2-SBD Docking Module by Accelrys Software
[52] in an attempt to find its probable natural substrate
(Table 2). According to phylogenetic analysis the Llbglu1
closely clustered withLotus japonicus b-glucosidases which
preferentially hydrolyse rhodiocyanoside A. So, docking
studies were carried out with rhodiocyanoside A as ligand,
into the modeled Leucaena b-glucosidase. Apart from this,
other flavonoids/isoflavonoid were also docked to know the
comparative binding affinities. The docked conformations
were ranked according to their binding energies (U total in
kcal/mol). The docking energy values were calculated as the
sum of the electrostatic, van der Waals energies and the
flexibility of the ligand itself. Low docking energy indicates
high binding ability. Receptor-ligand interactions were
shown in Ligplot [53] which was generated through PDB-
Sum on ebi server (http://www.ebi.ac.uk/pdbsum).
Results and discussion
Cloning and phylogenetic analysis ofLlbglu1
The Llbglu1 gene from L. leucocepha was isolated by PCR
amplification using Rapid Amplication of cDNA Ends
(RACE) and had already been deposited in the NCBI
Genbank database (Accession No. EU328158) by us. The
full-length gene was amplified by using gene specific
primers, cloned and sequenced. The Llbglu1 sequence was
analyzed using bioinformatics tools. It displayed high
sequence homology with b-glucosidases belonging toGlycosyl hydrolase family 1, a functionally diverse family
[10]. The full-length cDNA has an open reading frame of
1521 nucleotides encoding 507 amino acids. NCBI
BLASTP search of the amino acid sequence showed that
Llbglu1 has significant identity; 77 % with Lotus japonicus
b-glucosidase D7 (ACD65511), 74 % with Lotus japonicus
b-glucosidase D2 (ACD65510), 73 % with Lotus japoni-
cus b-glucosidase D4 (ACD65509), 70 % with cyanogenic
b-glucosidase of Trifolium repens (CAA40057) and 67 %
with amygdalin hydrolase isoform AH I precursor of
Prunus serotina (AAA93234). On the basis of BLAST
search it can be hypothesized that the b-glucosidase from
L. leucocephala is probably involved in defense by
cleaving hydroxynitrile compounds.
The function and specificity of Llbglu1 can be predicted
in a better accuracy by constructing a phylogenetic tree
incorporating Llbglu1 sequence with the other GH 1
b-glucosidases involved in defense system. In the phyloge-
netic tree of defensive b-glucosidases, it was observed that
12 different hydroxynitrile cleaving b-glucosidases form
three isolated clusters (Fig. 1). The first two clusters are
belonging to eudicotyledons and get separated by isoflavo-
noid b-glucosidases and third one fall into monocotyledons.
All the above twelve hydroxynitrile b-glucosidases were
selected for MSA along with Llbglu1 to analyze sequence
similarities among them with emphasis on N-terminal
sequence motif. The characteristic N-terminal signature
sequence, specific for hydroxynitrile b-glucosidases can be
observed as F-X-F-G-[AT]-A-[ST]-[SA]-[SA]-[FY]-Q-X-
EG-[AGE] in the MSA (Fig. S1). Llbglu1 satisfies the
hydroxynitrile cleaving GH1 b-glucosidase family with the
signature sequence as FIFGTASASYQYEGA which is
observed between the residues 39 and 53. In general, for the
Table 2 Comparative docking results of various glycosides with
modeled Llbglu1
S.
No.
Glucoside
class
Substrate DG
(kcal/
mol)
1. Hydroxy-nitriles Amygdalin -5.06
2. Hydroxy-nitriles Rhodiocynocide A -5.52
3. Isoflavonoid Genistein 7-O-glucoside -4.92
4. Isoflavonoid Genistein 40-O-glucoside -4.61
5. Flavonoid Naringenin 7-O-glucoside -5.11
6. Flavonoid Apigenin 7-O-glycoside -4.54
7. Nitro-phenyl p-Nitrophenyl b-D-glucopyranoside -6.45
Mol Biol Rep
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whole GH 1 b-glucosidase family, the signature sequence
has been reported as F-X-[FYWM]-[GSTA]-X-[GSTA]-
X-[GSTA]-[GSTA]-[FYN]-X-E-X-[GSTA][10, 54]. Llb-
glu1 also contains several sequence and structural motifs
which are highly conserved among many GH 1 b-glucosi-dases such as NEP and ENG which are structurally
important for enzyme activity [55, 56].
Expression, biochemical characterization and substrate
determination of Llbglu1
The Llbglu1 was found to have a signal peptide of 21 amino
acids analyzed using program SignalP 3.0 (http://www.cbs.
dtu.dk/services/SignalP/). A 1458 bp sequence encoding the
mature protein of 486 residues (without signal sequence) was
PCR amplified and subcloned into a pET-28a (?) expression
vector to give rise to His6 tagged fusion protein and trans-
formed into E. coli BL21 (DE3) cells. The crude extracts of
recombinant Llbglu1 were subjected to chromatography usinga NiNTA affinity column. The resulting molecular weight of
recombinant Llbglu1 was found to be*55 kDa (Fig. 2). The
optimal pH and temperature of the recombinantb-glucosidase
were found to be 4.8 and 45 C respectively (Fig. 3). The
optimum activity in the acidic range has been reported for
recombinant b-glucosidases from Arabidopsis [11, 57] and
also from rice [58]. The assays of enzyme resistance to dif-
ferent pH indicate that the recombinant Llbglu1 can preserve
its activity in broad range of pH 4.09.0 (Fig. S2).
LjBGLU2
LjBGLU4
LjBGLU7
Llbglu1
TrCBG
DcDBGLU
GmICHG
PsAH1
PsPH5
PsPH1
PsPH4
MeLinamarase
HbLinamarase
VaVH
AtTGG2
AtTGG1
SaMYR
BjMYR1
BjMYR
BnBGLU106
RsRMB1
PcCBG
AsGlu1
AsGlu2
ScBxGlcGLU
ZmGlu1
SbDhr1
SbDhr2
100
65
87
100
92
59
100
77
100
95
61
100
58
100
99
99
100
68
44
94
95
62
76
49
Hydroxynitrile
glucosides
(I)
Isoflavonoids
glucosides
Hydroxynitrile
glucosides
(II)
Glucosinolates
Avenacosides
Benzoxazinoid
glucosides
Hydroxynitrile
glucosides
(III)
Coniferin
Eudicotyledons
Monocotyledons
Fig. 1 Phylogenetic analysis of selected plant b-glucosidases
involved in the bioactivation of defense compounds. The phyloge-
netic tree includes hydroxynitrile and isoflavonoid glucoside-cleaving
b-glucosidases from eudicotyledons, glucosinolate degrading myro-sinases (Brassicales), and selected b-glucosidases involved in the
bioactivation of defense compounds in monocotyledons. Lotus
japonicus LjBGLU2 ACD65510; Lotus japonicus LjBGLU4
ACD65509; Lotus japonicus LjBGLU7 ACD65511; Leucaena leu-
cocephala Llbglu1 ABY48758; Trifolium repens TrCBG 1CBG-A;
Dalbergia cochinchinensis DcDBGLU AAF04007; Glycine max
GmICHG BAF34333; Prunus serotinaamygdalin PsAH1
AAA93234; Prunus serotina PsPH5 AAL35324; Prunus serotina
PsPH1 AAA93032; Prunus serotina PsPH4 AAL39079; Hevea
brasiliensis HbLinamarase ABL01537; Manihot esculenta MeLina-
marase AAB22162; Vicia sativa VaVH ABD03937; Arabidopsis
thaliana AtTGG2 NP568479; Arabidopsis thaliana AtTGG1NP851077; Sinapis alba SaMYR 1MYRA; Brassica juncea BjMYR1
AAG54074; Brassica juncea BjMYR CAA11412; Brassica napus
BnBGLU106 CAA42775; Raphanus sativus RsRMB BAB17227;
Avena sativa AsGlu1 CAA55196; Avena sativa AsGlu2 AAD02839;
Secale cereale ScBxGlcGLU AAG00614; Zea mays ZmGlu1
NP001105454; Sorghum bicolor SbDhr1 AAC49177; Sorghum
bicolorCyanogenic beta-glucosidase dhurrinase-2 AAK49119; Pinus
contorta PcCBG AAC69619
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When different substrates such as various nitro-phenyl-
derived chromogenic substrates were tested for linkage
specificity (Table 1) it is found that the enzyme can
hydrolyze only b-glucosides but not a-glucosides. The
recombinant enzyme showed preference for glucose as the
glycone moiety, though it can also hydrolyze galactose
(Table 1). Hydrolysis of pNPGal by L. leucocephala
recombinant b-glucosidase is not unusual as it has been
frequently noted with other plant b-glucosidases also [11,
57, 58]. To find the glycone specificity, kinetic constants
like Km and Vmax were determined for these substratesusing MichaelisMenten curve and were found to be
38.59 lM and 0.8237 lM/mg/min respectively for
pNPGlc, whereas for pNPGal the values were observed as
1845 lM and 0.1037 lM/mg/min, respectively.
When purified recombinant Llbglu1 was incubated with
amygdalin (Cyanogenic b-glucoside) and various flavonoid
glycosides such as genistein 7-O-glycosides, genistein 40-
O-glycoside, apigenin 7-O-glycoside, naringenin 7-O-gly-
coside and kaempferol 3-O-glycoside as substrates, we
found that Llbglu1 exhibited glycosidase activity towards
amygdalin and flavonoid glycosides except kaempferol
3-O-glycosides. The reaction product of amygdalin wasanalyzed by using LCMS (Fig. 4H), which shows the
mass of unused substrate (Molecular mass 457.43) with the
majority of sodium ion (m/z 480.139 [M ? 23]?) and the
reaction products show decrease of 162 mass for removal
of single glucose molecule with majority of hydrogen ion
(m/z 296.93 [M ? 1]?) and decrease of 324 molecular
mass for removal of two glucose molecule with the
majority of sodium ion (m/z 154.90 [M ? 23]?) respec-
tively. Flavonoid glycoside produces apigenin (flavones)
from apigenin 7-O-glycoside, genistein (isoflavones) from
genistein 7-O-glycosides and genistein 40-O-glycoside, and
naringenin (flavanone) from naringenin 7-O-glycoside,
which were co-eluted with their standards in analytical
HPLC. When the reaction products of these glycosides
were analyzed by using HPLC, genistein 7-O-glycoside
and genistein 40-O-glycoside gave a peak that had the same
retention time (14.3) and the UV-spectra with genistein. On
the other hand, naringenin and apigenin reaction product
generate the peak that had the same retention time (11.2
and 12.3) and the UV spectra with naringenin and apigenin
respectively (Fig. 4(AG)). These results give the qualita-
tive information that Llbglu1 can hydrolyse natural sub-
strates of hydroxynitrile glycosides and flavonoid/
isoflavonoid glycosides.
Homology modeling and active site identification
The primary amino acid sequence of Llbglu1 was searched
against PDB which showed the high percentage identity:
70 % with cyanogenic b-glucosidase from Trifolium repens
(1CBG), 53 % with Strictosidine glucosidase (2JF7:A) from
Rauvolfia serpentina, 49 % with Dhurrinase (1V02:E) from
Fig. 2 SDS-PAGE analysis of the purified recombinant Llbglu1.
Lane 1, purified recombinant Llbglu1, Lane 2 protein marker, broad
range (7175 kDa)
A
B
Fig. 3 The Effects of pH (A) and temperature (B) on the activity of
the recombinant b-glucosidase activity. b-glucosidase activity was
assayed at various pH in 0.1 mM citratephosphate buffer (3.56.0),
phosphate buffer (6.57.0). b-glucosidase activity was assayed at
different temperatures. Activity is expressed as a percentage of the
maximum activity
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Sorghum bicolor and 47 % with Myrosinase (1MYR:A)
from Sinapis alba. Out of 507 residues of Llbglu1 submitted
for homology modeling, 482 residues were modeled in the
3D structure. 25 residues at the N-terminal end remained
unmodeled because they are not having regular secondary
structures and might come in flexible loop region. In this
model a conserved (b/a)8 barrel was observed (Fig. 5A)
which is a common feature among the family 1 b-glucosi-
dase belonging to clan GH-A. The geometry of the final
refined model was evaluated with Ramachandrans plot (Fig.
S3). From the pairwise structural alignment, it is quite evi-
dent that the contents of secondary structural elements in
Llbglu1 are more or less similar to 1CBG (Fig. S4). The
secondary structure of Llbglu1 contains 19 a-helices and 17
b-sheets respectively.Due to presence of TIM fold, locations
of secondary structures in Llbglu1 are highly conserved and
matching exactly with template 1CBG; hence their biolog-
ical functions may be quite similar.
The structural superposition of template 1CBG and
Llbglu1 (Fig. 5B) shows that the amino acids in the active
site are conserved. A good number of 3D crystal structures
of GH1 enzymes helped to establish the link between
active site residues and ligand components for the hydro-
lysis mechanism. b-glucosidase ofZea mays has a slot-like
active site, with the catalytic proton donor/base and
nucleophile being Glu 191 and Glu 406 respectively [34].
In Trifolium cyanogenic b-glucosidase, those catalytic
residues are Glu 183 and Glu 397 corresponds Glu 199 and
Glu 413 in Llbglu1 (Fig. 5C). Other important residues in
the active site of Llbglu1 are His 153 (137), Asn 198 (182),
Val 270 (254), Asn 340 (324), and Trp 462 (446) (corre-
sponding residues of 1CBG are written in brackets). Most
of the active site residues lie on the loops of the TIM barrel
fold and these residues are involved in glycone binding
pocket. The catalytic site residues are highly conserved in
GH 1 family and these are also present in Llbglu (Fig. S5).
Fig. 4 HPLC chromatogram of Llbglu1 assay mixture with A Gen-istein 7-O-glucoside and B genistein 40-O-glucoside, C Std1: geni-
stein, D naringenin 7-O-glucoside, E Std2: naringenin, F apigenin
7-O-glucoside, G Std3: apigenin, H LCMS: positive ion massspectrum of amygdalin. P1, P2 and P3 are genistein, naringenin and
apigenin respectively
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Molecular docking of rhodiocyanoside A and other
classes of glucosides
Phylogenetic analysis ofLeucaena b-glucosidase (Llbglu1)
shows that it is closely related to Lotus japonicus b-glu-
cosidases which hydrolyze the rhodiocyanoside A prefer-
entially [28]. However, to our knowledge, there are no
reports on the occurrence of hydroxynitrile glycosides in
Leucaena leucocephala (subabul). Therefore, docking
studies were carried out with rhodiocyanoside A into the
modeled Llbglu1 in an attempt to find its probable natural
substrate. Docking of the rhodiocyanoside A in the activesite of Llbglu1 (Fig. 5D) showed that it binds to the
enzyme pocket with high affinity. The free energy change
(DG) of the best pose of enzyme-ligand complex was found
as -5.52 kcal/mol.
Many variations of amino acid residues occur with dif-
ferent GH 1 members, but they have very similar active site
structures to ensure that their analogous residues will have
most of the same interactions. In general, those glycosyl
ligand that are free to take up different ring conformations
on binding in active sites of GH members are found as
relaxed 4C1 conformers [59]. Deeper in the cleft it has
glycon-binding region with Glu 199 and Glu 413 interacting
with the O2 atom of the glycon glucosyl residue. The dis-
tance of O2 were found 3.0 A and 4.2 A from the Glu 199
and Glu 413 side chain terminal oxygen respectively
(Fig. 6B). These distances clearly show that one water
molecule can come and hydrolyze the glycosidic bond by
following the well known acid/base catalysis mechanism.
Three hydrogen bonds are clearly visible, O3 and O4 of
ligand are acting as hydrogen bond acceptor, whereas NE1
(Trp 470) and NE2 (Gln 49) act as hydrogen bond donor.Aglycon moiety of the ligand (N7, H-bond donor) forms
hydrogen bond with Thr 202 (OG1, H-bond acceptor). Total
17 residues were found in 5 A vicinity of the docked ligand
in Llbglu1 pocket (Fig. 6D). These are Gln 49, His 153, Trp
154, Asn198, Glu 199, Trp 201, Thr 202, Val 270, His 272,
Met 294, Tyr 342, Trp 385, Glu 413, Trp 462, Glu 469, Trp
470 and Phe 478. Out of 17 residues, 9 are aromatic ring
containing amino acids (W-5, H-2, F-1 and Y-1). Definitely
these aromatic rings containing amino acid have important
Fig. 5 A Modeled Llbglu1:
a-helices, b-sheets and loops are
shown in red, yellow and greencolor respectively.
B Superimposition of modeled
Llbglu1 (red) with Trifolium
1CBG (green). C Top view of
the barrel showing the
superimposition of active site
residues of Llbglu1 with
Trifolium 1CBG. 1CBG and
Llbglu1 are shown in green and
red colors respectively whereas
active site residues are
represented in line form are of
modeled Llbglu1. D The surface
structure of the Llbglu1 with the
docked Rhodiocyanoside A
(Carbongreen, oxygenred,
nitrogenblue). (Color figure
online)
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role to attract and hold the substrate till the end of the
hydrolysis reaction. Tryptophan residue (Trp 385) of Llb-
glu1 is conserved within the GH 1 family and its role in
substrate recognition has been described previously [35].
The Trp 385 in all known plant glucosidase shows its side
chain torsion angle v * 60. The residues Trp 462 and Glu
469 in Llbglu1 are also conserved in the active pocket of
other GH 1 members [60].
Fig. 6 Interactions of catalytic residues of Llbglu1 with Rhodiocy-
anoside A through ligand flexible fit docking; A Ligplot: schematic
diagrams of Llbglu1-Rhodiocyanoside A (proteinligand) interac-tions. Hydrogen bonds are indicated by dashed lines between the
atoms involved, while hydrophobic contacts are represented by an arc
with spokes radiating towards the ligand atoms they contact. The
contacted atoms are shown with spokes radiating back. B Three
dimensional orientations of acid/base catalytic residues Glu 199 and
Glu 413 (green) in binding site of Llbglu1 along with the substrate
Rhodiocyanoside A (yellow). The distances of glycosidic oxygen of
Rhodiocyanoside A with side chain oxygen of catalytic Glu-199 and
Glu-413 are 3.0 and 4.2 A
, Aglucon nitrogen in Rhodiocyanoside A(N7) forms hydrogen bonding with Thr 202 ( green). C Molecular
surface structure of the residues lining the active site pocket of the
Llbglu1 enzyme with Rhodiocyanoside A (ball and stick represen-
tation) positioned in the binding cleft. D Locations of all the 17
residues (stick representation) forming the binding pocket containing
docked Rhodiocyanoside A ligand. (Color figure online)
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All the other glycosides that were hydrolysed by Llb-
glu1, reported in the study, were docked into the 3D model
of the enzyme to get a plausible docking arrangement. The
free energy (DG) of binding for these substrates were
presented in Table 2. Due to wide active site pocket
present in the enzyme, a range of substrates are possible for
the hydrolysis. However, the mode of binding is primarily
governed by the aglycone moiety as described for the
rhodiocyanoside A. DG for the binding of amygdalin
(-5.06 kcal/mol) shows that the first choice of the substrates
for the enzyme may belong to a class of hydroxynitrile
glucosides. However, flavonoid/isoflavonoid glucosides
were showing a binding energy range of 5.114.54 kcal/mol
which suggest to consider them too as good substrates for the
enzyme. All the biochemically tested substrates when
docked into the same active site pocket, perfectly fit into the
catalytic pocket and depicting the plausible arrangement
surrounded by active site residues (Fig. 7).
In conclusion, our results suggest that Llbglu1 is a Gly-
cosyl hydrolase family 1b-glucosidase. Phylogenetic analysis
shows that thisb-glucosidase is involved in defense, probably
by hydrolyzing hydroxynitrile glucosides. Sequence of the
mature b-glucosidase was expressed in E. coli in active form.
It has a pH and temperature optima of 4.8 and 45 C
respectively. The enzyme is stable in pH range 4.09.0 and
has a preference for glucose as glycone moiety. These prop-
erties of L. leucocephala b-glucosidase are in broad agree-
ment with other plant b-glucosidases. The enzyme readily
hydrolyzed a hydroxynitrile glycoside, amygdalin. Further, it
also shows hydrolyzing activity towards flavonoid/isoflavo-
noid glucosides. Structural analysis of the modeled Llbglu1
showed that most of the active site residues are conserved and
molecular docking analysis revealed that rhodiocyanoside A
could be a preferred substrate for the enzyme. Functional
characterization and structural analysis of Llbglu1 will pave
the way for the identification of its natural substrate in vivo
and may find application in genetic engineering of the crop
plants with resistance to insect and pathogens.
Acknowledgments Financial support in the form of Junior & Senior
Research Fellowships to Noor M. Shaik by Council of Scientific andIndustrial Research, New Delhi and to Anurag Misra by University
Grants Commission, New Delhi is gratefully acknowledged.
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