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

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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

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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

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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

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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

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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.

References

1. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V,

Henrissat B (2009) The carbohydrate-active enzymes database

(CAZy): an expert resource for glycogenomics. Nucleic Acids

Res 37:233238

Fig. 7 Docked complexes of modeled Llbglu1 with various gluco-

sides. A Amygdalin, B rhodiocyanoside A, C genistein 7-O-gluco-

side, D genistein 40-O-glucoside, E Naringenin 7-O-glucoside,

F apigenin 7-O-glycoside, G p-Nitrophenyl b-D-glucopyranoside,

H all the substrates in the same pocket

Mol Biol Rep

123

7/28/2019 10b-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides

12/13

2. Morant AV, Jrgensen K, Jrgensen C, Paquette SM, Sanchez-

Perez R, Mller BL, Bak S (2008) b-Glucosidases as detonators

of plant chemical defense. Phytochemistry 69:17951813

3. Halkier BA, Gershenzon J (2006) Biology and biochemistry of

glucosinolates. Annu Rev Plant Biol 57:303333

4. Suzuki H, Takahashi S, Watanabe R, Fukushima Y, Fujita N,

Noguchi A, Yokoyama R, Nishitani K, Nishino T, Nakayama T

(2006) An isoflavone conjugate-hydrolyzing b-glucosidase from

the roots of soybean (Glycine max) seedlings: purification, gene

cloning, phylogenetics, and cellular localization. J Biol Chem

281:3025130259

5. Nisius A (1988) The stromacentre in avena plastids: an aggre-

gation ofb-glucosidase responsible for the activation of oat-leaf

saponins. Planta 173:474481

6. Poulton JE (1990) Cyanogenesis in plants. Plant Physiol

94:401405

7. Leah R, Kigel J, Svendsen I, Mundy J (1995) Biochemical and

molecular characterization of a barley seed b-glucosidase. J Biol

Chem 270:1578915797

8. Kristoffersen P, Brzobohaty B, Hohfeld I, Bako L, Melkonian M,

Palme K (2000) Developmental regulation of the maize Zm-

g60.1 gene encoding a b-glucosidase located to plastids. Planta

210:407415

9. Lee KH, Piao HL, Kim HY, Choi SM, Jiang F, Hartung W,

Hwang I, Kwak JM, Lee IJ, Hwang I (2006) Activation of glu-

cosidase via stress-induced polymerization rapidly increases

active pools of abscisic acid. Cell 126:11091120

10. Dharmawardhana DP, Ellis BE, Carlson JE (1995) A b-glucosi-

dase from lodgepole pine xylem specific for the lignin precursor

coniferin. Plant Physiol 107:331339

11. Escamilla-Trevino LL, Chen W, Card ML, Shih M, Cheng CL,

Poulton JE (2006) Arabidopsis thaliana b-glucosidases BGLU45

and BGLU46 hydrolyse monolignol glucosides. Phytochemistry

67:16511660

12. Mizutani M, Nakanishi H, Ema J, Ma S, Noguchi E, Inohara-

Ochiai M, Fukuchi-Mizutani M, Nakao M, Sakata K (2002)

Cloning of betaprimeve-rosidase from tea leaves, a key enzyme

in tea aroma formation. Plant Physiol 130:21642176

13. Fia G, Giovani G, Rosi I (2005) Study of beta-glucosidase pro-

duction by wine-related yeasts during alcoholic fermentation: a

new rapid fluorimetric method to determine enzymatic activity.

J Appl Microbiol 99:509517

14. Maicas S, Mateo J (2005) Hydrolysis of terpenyl glycosides in

grape juice and other fruit juices, A review. Appl Microbiol

Biotechnol 67:322335

15. Sue M, Ishihara A, Iwamura H (2000) Purification and charac-

terization of a b-glucosidase from rye (Secale cereale L.) seed-

lings. Plant Sci 155:6774

16. Sue M, Ishihara A, Iwamura H (2000) Purification and charac-

terization of a hydroxamic acid glucoside b-glucosidase from

wheat (Triticum aestivum L.) seedlings. Planta 210:432438

17. Cairns JRK, Champattanachai V, Srisomsap C, Wittman-Liebold

B, Thiede B, Svasti J (2000) Sequence and expression of thai

rosewood b-glucosidase/b-fucosidase, a family 1 glycosylhydrolase glycoprotein. J Biochem 128:9991008

18. Niemeyer HM (1988) Hydroxamic acids (4-hydroxy-1,4-benz-

oxazin-3-ones), defense chemicals in the gramineae. Phyto-

chemistry 27:33493358

19. Daniels LB, Coyle PJ, Chiao YB, Glew RH, Labow RS (1981)

Purification and characterization of a cytosolic broad specificity

beta-glucosidase from human liver. J Biol Chem 256:1300413013

20. Berrin J, McLauchlan WR, Needs P, Williamson G, Puigserver

A, Kroon PA, Juge N (2002) Functional expression of human

liver cytosolic b-glucosidase in Pichia pastoris: insight into its

role in the metabolism of dietary glucosides. Eur J Biochem

269:249258

21. Chiou T, Lin Y, Su N, Lee M (2010) b-Glucosidase isolated from

soybean okara shows specificity toward glucosyl isoflavones.

J Agric Food Chem 58(15):88728878

22. Lechtenberg M, Nahrstedt A (1999) Cyanogenic glycosides,

naturally occurring glycosides. Wiley, Chichester, pp 147191

23. Bjarnholt N, Rook F, Motawia MS, Cornett C, Jrgensen C,

Olsen CE, Jaroszewski J, Bak S, Mller BL (2008) Diversifica-

tion of an ancient theme: hydroxynitrile glucosides. Phytochem-

istry 69:15071516

24. Oxtoby E, Dunn MA, Pancoro A, Hughes MA (1991) Nucleotide

and derived amino acid sequence of cyanogenic b-glucosidase

(linamarase) from white clover (Trifolium repens L.). Plant Mol

Biol 17:209219

25. Hughes MA, Brown K, Pancoro A, Murray BS, Oxtoby E,

Hughes J (1992) A molecular and biochemical analysis of the

structure of the cyanogenic b-glucosidase from cassava (Manihot

esculenta Crantz). Arch Biochem Biophys 295:273279

26. Zhou J, Hartmann S, Shepherd BK, Poulton JE (2002) Investi-

gation of the microheterogeneity and aglycone specificity-con-

ferring residues of black-cherry prunasin hydrolases. Plant

Physiol 129:12521264

27. Ahn Y, Saino H, Mizutani M, Shimizu B, SakataK (2007)Vicianin

hydrolase is a novel cyanogenic b-glucosidase specific to b-vici-

anoside (6-O-a-L-arabinopyranosyl-b-D-glucopyranoside) in seeds

ofVicia angustifolia. Plant Cell Physiol 48:938947

28. Morant AV, Bjarnholt N, Kragh ME, Kjaergaard CH, Jrgensen

K, Paquette SM, Piotrowski M, Imberty A, Olsen CE, Mller BL,

Bak S (2008) The b-glucosidases responsible for bioactivation of

hydroxynitrile glucosides in Lotus japonicus. Plant Physiol

147:10721091

29. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon J, Davies G

(1995) Conserved catalytic machinery and the prediction of a

common fold for several families of glycosyl hydrolases. Proc

Natl Acad Sci USA 92:70907094

30. Jenkins J, Leggio LL, Harris G, Pickersqill R (1995) b-Glucosi-

dase, b-galactosidase, family A cellulases, family F xylanases

and two barley glycanases form a superfamily of enzymes wit

8-fold b/a architecture and with two conserved glutamates near

the carboxy-terminal ends ofb-strands four and seven. FEBS let

362:281285

31. Tribolo S, Berrin J, Kroon PA, Czjzek M, Juge N (2007) The

crystal structure of human cytosolic b-glucosidase unravels the

substrate aglycone specificity of a family 1 glycoside hydrolase.

J Mol Biol 370:964975

32. Berrin JG, Czjzek M, Kroon PA, McLauchlan WR, Puigserver A,

Williamson G, Juge N (2003) Substrate (aglycone) specificity of

human cytosolic beta-glucosidase. Biochem J 373:4148

33. Cicek M, Blanchard D, Bevan DR, Esen A (2002) The aglycone

specificity-determining sites are different in 2,4-dihydroxy-7-

methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (maize

b-glucosidase) and dhurrinase (sorghum b-glucosidase). J Biol

Chem 275:2000220011

34. Czjzek M, Cicek M, Zamboni V, Bevan DR, Henrissat B, Esen A

(2000) The mechanism of substrate (aglycone) specificity in beta-glucosidases is revealed by crystal structures of mutant maize

beta-glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin com-

plexes. Proc Natl Acad Sci USA 97:1355513560

35. Czjzek M, Cicek M, Zamboni V, Burmeister WP, Bevan DR,

Henrissat B, Esen A (2001) Crystal structure of a monocotyledon

(maize ZMGlu1) beta-glucosidase and a model of its complex

with p-nitrophenyl beta-D-thioglucoside. Biochem J 354:

3746

36. Verdoucq L, Moriniere J, Bevan DR, Esen A, Vasella A, Hen-

rissat B, Czjze M (2004) Structural determinants of substrate

specificity in family 1 beta-glucosidases: novel insights from the

crystal structure of sorghum dhurrinase-1, a plant beta-

Mol Biol Rep

123

7/28/2019 10b-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides

13/13

glucosidase with strict specificity, in complex with its natural

substrate. J Biol Chem 279:3179631803

37. Verdoucq L, Czjzek M, Moriniere J, Bevan DR, Esen A (2003)

Mutational and structural analysis of aglycone specificity in maize

and sorghum beta-glucosidases. J Biol Chem 278:2505525062

38. Morant AV, Jrgensen K, Jrgensen B, Dam W, Olsen CE,

Mller BL, Bak S (2007) Lessons learned from metabolic engi-

neering of cyanogenic glucosides. Metabolomics 3:383398

39. Gatehouse JA (2008) Biotechnological prospects for engineering

insect-resistant plants. Plant Physiol 146:881887

40. Nielsen KA, Hrmova M, Nielsen JN, Forslund K, Ebert S, Olsen

CE, Fincher GB, Mller BL (2006) Reconstitution of cyano-

genesis in barley (Hordeum vulgare L.) and its implications for

resistance against the barley powdery mildew fungus. Planta

223:10101023

41. Tattersall DB, Bak S, Jones PR, Olsen CE, Nielsen JK, Hansen

ML, Hj PB, Mller BL (2001) Resistance to an herbivore

through engineered cyanogenic glucoside synthesis. Science

293:18261828

42. Rastogi S, Dwivedi UN (2003) Agrobacterium tumefaciense-

mediated transformation of Leucaena leucocephalaa multi-

purpose tree legume. Physiol Mol Biolplants 9:207216

43. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular

evolutionary genetic analysis (MEGA) software version 4.0. Mol

Biol Evol 24:15961599

44. Ko JH, Kim BG, Hur HG, Lim Y, Ahn JH (2006) Molecular

cloning, expression and characterization of a glycosyltransferase

from rice. Plant Cell Rep 25:741746

45. Marti-Renom M, Stuart A, Fiser A, Sanchez R, Melo F, Sali A

(2000) Comparative protein structure modeling of genes and

genomes. Annu Rev Biophys Biomol Struct 29:291325

46. DeLano W (2002) The pymol users manual. DeLano Scientific,

San Carlos

47. Laskowski R, McArthur M, Moss D, Thornton J (1993) PRO-

CHECK: aprogram to check the stereo-chemical quality of pro-

tein structures. J Applied Cryst 26:283291

48. Goute P, Courcelle E, Stuart D, Metoz F (1999) ESPript:analysis

of multiple sequence alignments in PostScript. Bioinformatics

15:305308

49. Shindyalov I, Bourne P (1998) Protein structure alignment by

incremental combinatorial extension (CE) of the optimal path.

Protein Eng 11:739747

50. Guda C, Scheeff E, Bourne P, Shindyalov I (2001) A new

algorithm for the alignment of multiple protein structures using

Monte Carlo optimization. Proc Pac Sym Biocomput 6:275286

51. Guda C, Lu S, Scheeff E, Bourne P, Shindyalov I (2004) CE-MC:

a multiple protein structure alignment server. Nucleic Acids Res

32:100103

52. Accelrys (2003) Structure-based drug design with discovery stu-

dio, Accelrys, version 0406. Accelrys Software Inc., San Diego

53. Wallace A, Laskowski R, Thornton J (1995) LIGPLOT: a pro-

gram to generate schematic diagrams of protein-ligand interac-

tions. Prot Eng 8:127134

54. Henrissat B (1991) A classification of glycosyl hydrolases based

on amino acid sequence similarities. Biochem J 280:309316

55. Baird SD, Hefford MA, Johnson DA, Sung WL, Yaguchi M,

Seligy VL (1990) The Glu residue in the conserved Asn-Glu-Pro

sequence of two highly divergent endo-b-1,4-glucanases is

essential for enzymatic activity. Biochem Biophys Res Commun

169:10351039

56. Withers SG, Warren RAJ, Street IP, Rupitz JB, Kempton JB,

Aebersold R (1990) Unequivocal demonstration of the involve-

ment of a glutamate residue as a nucleophile in the mechanism of

a retaining glycosidase. J Am Chem Soc 112:58875889

57. Xu Z, Escamilla-Trevino LL, Zeng L, Lalgondar M, Bevan DR,

Winkel BSJ, Mohamed A, Cheng CL, Shih MC, Poulton JE, Esen

A (2004) Functional genomic analysis of Arabidopsis thaliana

glycoside hydrolase family 1. Plant Mol Biol 55:343367

58. Opassiri R, Pomthong B, Onkoksoong T, Akiyama T, Esen A,

Cairns JRK (2006) Analysis of rice glycosyl hydrolase family 1

and expression of Os4bglu12 b-glucosidase. BMC Plant Biol

6:33

59. Hill AD, Reilly PJ (2008) Computational analysis of glycoside

hydrolase family 1 specificities. Biopolymers 89(11):10211031

60. Barleben L, Panjikar S, Ruppert M, Koepke J, Stockigt J (2007)

Molecular architecture of strictosidine glucosidase: the gateway

to the biosynthesis of the monoterpenoidIndole alkaloid family.

Plant Cell 19:28862897

Mol Biol Rep

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