Mol Biol Rep DOI 10.

1007/s11033-012-2179-6

Functional characterization, homology modeling and docking studies of b-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from 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 in plants including defense, whereby hydrolyzing the defensive 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 puried by Ni NTA afnity chromatography. The optimal temperature and pH for this b-glucosidase were found to be 45 C and 4.8, respectively. The puried 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 avonoid and isoavonoid 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 nd 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,
Electronic supplementary material The online version of this article (doi:10.1007/s11033-012-2179-6) contains supplementary material, 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: bm.khan@ncl.res.in A. Misra S. Ramakumar Department of Physics, Bioinformatics Centre, Indian Institute of Science, Bangalore 560012, India

Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted 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 carbohydrates or between a carbohydrate and non-carbohydrate moiety. Based on the sequence similarities, these enzymes have been classied into 115 families, whose unique features and representative members are described in the Carbohydrate-Active enzymes database (http://www.cazy.org/) [1]. Plant b-glucosidases belonging to Glycosyl hydrolase family 1 (GH 1), serve a number of diverse and important functions, including bioactivation of defense compounds [26], cell wall degradation in endosperm during germination [7], activation of phytohormones [8, 9] and lignications [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

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compounds are often stored as b-glucosides and bio-activated by specic 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 naphthoquinones 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], isoavonoid glycosides in legumes [17] and glucosinolates mainly in brassicales [3]. The physiological functions of b-glucosidases varies greatly depending upon their origin (plants, fungi, animals or bacteria) and substrate specicity. A distinguished feature of the human cytostolic b-glucosidase (hCBG) is its ability to hydrolyze many common dietary xenobiotics, including glycosides of phytoestrogens, avonoids, simple phenolics and cyanogens [19, 20]. The hCBG shows high specicity for 40 - and 7-glucosides of isoavones, avonols, avones and avonones, but does not hydrolyze 3-linked avonoid glucosides [20]. A b-glucosidase from soybean and okara shows the specicity towards glucosyl isoavones [21]. Among the plants harboring cyanogenic glycosides, several of them produce b- and c-hydroxynitriles also. Because of the striking structural similarities among a-, b-, and c-hydroxynitriles and a high frequency of co-occurrence, it has been proposed that the compounds are biosynthetically related to each other [22, 23]. These hydroxynitrile glycosides are bioactivated by specic b-glucosidases. The hydroxynitrile cleaving b-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 structures, 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 catalytic 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 rather broad range of glycone specicity, however, one enzyme may hydrolyze b-D-glucosides, b-D-galactosides, b-D-fucosides, b-D-mannosides and a-L-arabinosides, or may be specic 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 specicity differences that determine their natural substrate. Structures of complexes of enzymes with inhibitors, and mutant enzymes with substrates, along with mutagenesis and chimera studies comparing similar enzymes with divergent specicities, have suggested that the basis of aglycone specicity 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 specicity. Maize ZmGlu1 and Sorghum dhurrinase 1 (SbDhr1) are closely related, displaying 70 % amino acid sequence identity, but have distinct specicities. ZmGlu1 has broad range specicity, but cannot hydrolyze dhurrin, the natural substrate of SbDhr1, while SbDhr1 hydrolyzes only dhurrin. Studies of reciprocal ZmGlu1/SbDhr1 chimeric enzymes [33] and subsequent structural and site-directed mutagenesis studies [34 37] indicated that aglycone specicity determining sites are different in ZmGlu1 and SbDhr1. Due to its unique genetic simplicity, the cyanogenic glycoside 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 availability of the genes encoding the biosynthetic enzymes of secondary metabolism has made the transfer of entire biosynthetic pathways between plants feasible [41]. Therefore, identication and characterization of genes involved in biosynthesis and bio-activation of hydroxynitrile compounds not only dene structure and function of the enzymes, but also can nd 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 of Llbglu1 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 nd 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 amplication was purchased from Sigma USA. All restriction enzymes and T4 DNA ligase were used from Promega, USA. pGEM-T Easy vector (Promega, USA) used for cloning of PCR amplied products and pET-28a(?) (Novagen, USA) for expression of protein. NiNTA agarose afnity column (Qiagen, USA) was used for protein purication. All the substrates used for enzyme assay were obtained from Sigma, USA, unless otherwise specied. Cloning of full length cDNA of GH1 b-glucosidase Total RNA was isolated from one-week old in vitro grown seedlings of L. leucocephala using the TRIZOL reagent and the rst strand cDNA was synthesized by using BD powerscript reverse transcriptase. PCR amplication was performed with specic primers designed from Llbglu1 sequence having restriction sites for KpnI at forward primer 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). Specic primer sequences used for Llbglu1 were 50 -GGTACCATGATGAAGAAGG TGATGGTAGTA-30 (sense) and 50 -CTCGAGTTAATAT TTTTGAAGGAAGTTCCTG-30 (antisense), where the underlined sequences are the restriction sites for KpnI and XhoI, respectively. PCR amplication 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 puried with Gen EluteTM gel extraction kit (Sigma, USA) and subcloned into pGEM-T Easy vector (Promega, USA) and conrmed 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 constructed using Mega 4.0 [43] with 1000 bootstrap trials.

To study sequence similarities of Llbglu1 with 12 different 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 gure was generated using ESPript 2.2 server (http://espript.ibcp.fr/ESPript). The protein sequences used for MSA are LjBGLU2, Lotus japonicus b-glucosidase 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 glucosidase (GenBank Accession No. ABL01537); MeLinamarase, 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 purication 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 amplied with AccuTaq-LA DNA polymerase by using primers containing EcoRI at forward primer 50 -GAATTCGATGCAAC AAATGATATTTCC-30 and NotI at reverse primer 50 GCGGCCGCTTAATATTTTTGAAGGAAGTTCCTG-30 . 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 A600 reached 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 purication with Ni NTA agarose afnity column (Qiagen, USA). The binding of b-glucosidase to the NiNTA agarose beads was carried out at pH 8.0 in 10 mM Tris buffer and washing of nonspecic proteins at pH 6.3 in 50 mM citratephosphate buffer and elution of the enzyme at pH 4.5 in 50 mM citratephosphate buffers. The puried fractions were analyzed 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). Appropriately diluted enzyme was incubated with substrate (1 mM pNPGlc) in 50 mM citratephosphate buffer (pH 4.8) in a nal 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-efcient of 1.77x104. One unit of the enzyme is dened as the amount of enzyme that liberates 1 lmol of p-nitrophenol/min under the assay conditions. For the avonoid glycosides the reaction mixture contained 20 lg of puried Llbglu1, 100 mM TrisHCl (pH 4.8) and 70 lM substrates. The avonoid glycosides were purchased from Chromadex (www.chromadex.com) and
Table 1 Activity of the puried recombinant b-glucosidase with various nitro-phenyl derived chromogenic substrates S.No 1 2 3 4 5 6 7 8 9 10 11
a

standards were purchased from Sigma Aldrich (Sigma, USA). The reaction mixture was incubated at 45 C for 1 h and the reaction mixtures of avonoid 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 reaction product was analyzed by high performance liquid chromatography (HPLC, Perkin Elmer, USA) equipped with a diode array detector (DAD) and a Waters symmetry C18 column (5 lm particle size, 4.6 mm 9 25 cm, supelco analytical, Sigma, USA). For generation of an analytical scale, the mobile phase was consisted of sterile milliQ water and 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 ow 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 puried 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. Temperature 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 modeling based on high resolution crystal structure of homologous protein. To nd 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 white clover (Trifolium repens, PDB: 1CBG) was found closest

Substrate p-Nitrophenyl b-D-glucopyranoside p-Nitrophenyl b-D-glucoronide p-Nitrophenyl-N-acetyl-1-thio-bglucosaminide p-Nitrophenyl a-D-glucopyranoside p-Nitrophenyl N-acetyl-b-D-glucosaminide p-Nitrophenyl b-D-galactopyranoside p-Nitrophenyl b-D-mannopyranoside p-Nitrophenyl b-D-xylopyranoside p-Nitrophenyl b-L-arabinopyranoside o-Nitrophenyl b-D-glucopyranoside p-Nitrophenyl N-acetyl-a-D-glucosaminide

Relative activitya (%) 100 0.8 0.5 0.1 4.0 53.0 2.2 0.6 6.0 42.0 0.4

The puried b-glucosidase was incubated at optimum pH (4.8) with potential substrates provided at 5 mM nal 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 secondary structural elements (a-helices, b-sheets and turns) of Llbglu1 with that of 1CBG, the pair wise sequence alignment of these 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 combinatorial 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 of plant 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 nd its probable natural substrate (Table 2). According to phylogenetic analysis the Llbglu1 closely clustered with Lotus 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 avonoids/isoavonoid were also docked to know the comparative binding afnities. 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 exibility of the ligand itself. Low docking energy indicates high binding ability. Receptor-ligand interactions were shown in Ligplot [53] which was generated through PDBSum on ebi server (http://www.ebi.ac.uk/pdbsum).

Table 2 Comparative docking results of various glycosides with modeled Llbglu1 S. No. 1. 2. 3. 4. 5. 6. 7. Glucoside class Hydroxy-nitriles Hydroxy-nitriles Isoavonoid Isoavonoid Flavonoid Flavonoid Nitro-phenyl Substrate DG (kcal/ mol) -5.06 -5.52 -4.92 -4.61 -5.11 -4.54 -6.45

Amygdalin Rhodiocynocide A Genistein 7-O-glucoside Genistein 40 -O-glucoside Naringenin 7-O-glucoside Apigenin 7-O-glycoside p-Nitrophenyl b-D-glucopyranoside

Results and discussion Cloning and phylogenetic analysis of Llbglu1 The Llbglu1 gene from L. leucocepha was isolated by PCR amplication 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 amplied by using gene specic primers, cloned and sequenced. The Llbglu1 sequence was analyzed using bioinformatics tools. It displayed high sequence homology with b-glucosidases belonging to Glycosyl 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 signicant identity; 77 % with Lotus japonicus b-glucosidase D7 (ACD65511), 74 % with Lotus japonicus b-glucosidase D2 (ACD65510), 73 % with Lotus japonicus 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 specicity 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 phylogenetic tree of defensive b-glucosidases, it was observed that 12 different hydroxynitrile cleaving b-glucosidases form three isolated clusters (Fig. 1). The rst two clusters are belonging to eudicotyledons and get separated by isoavonoid 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, specic for hydroxynitrile b-glucosidases can be observed as F-X-F-G-[AT]-A-[ST]-[SA]-[SA]-[FY]-Q-XEG-[AGE] in the MSA (Fig. S1). Llbglu1 satises 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

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99 99 94 49 76 62 95 100 100 58

LjBGLU2 LjBGLU4 LjBGLU7 Llbglu1 TrCBG DcDBGLU GmICHG PsAH1 PsPH5 PsPH1 PsPH4 MeLinamarase HbLinamarase VaVH
Hydroxynitrile glucosides Isoflavonoids glucosides Hydroxynitrile glucosides

(I)

Eudicotyledons

100

(II)

44 100 92 100 87

59

AtTGG2 AtTGG1 SaMYR BjMYR1 BjMYR BnBGLU106 RsRMB1 PcCBG

Coniferin Glucosinolates

65

68 61 100 95

100

AsGlu1 AsGlu2 ScBxGlcGLU ZmGlu1 SbDhr1 SbDhr2

Avenacosides Benzoxazinoid glucosides Hydroxynitrile glucosides

Monocotyledons

77

(III)

Fig. 1 Phylogenetic analysis of selected plant b-glucosidases involved in the bioactivation of defense compounds. The phylogenetic tree includes hydroxynitrile and isoavonoid glucoside-cleaving b-glucosidases from eudicotyledons, glucosinolate degrading myrosinases (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 leucocephala 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 MeLinamarase AAB22162; Vicia sativa VaVH ABD03937; Arabidopsis thaliana AtTGG2 NP568479; Arabidopsis thaliana AtTGG1 NP851077; 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 bicolor Cyanogenic beta-glucosidase dhurrinase-2 AAK49119; Pinus contorta PcCBG AAC69619

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]. Llbglu1 also contains several sequence and structural motifs which are highly conserved among many GH 1 b-glucosidases 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 amplied and subcloned into a pET-28a (?) expression vector to give rise to His6 tagged fusion protein and transformed into E. coli BL21 (DE3) cells. The crude extracts of recombinant Llbglu1 were subjected to chromatography using a NiNTA afnity column. The resulting molecular weight of recombinant Llbglu1 was found to be *55 kDa (Fig. 2). The optimal pH and temperature of the recombinant b-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 different pH indicate that the recombinant Llbglu1 can preserve its activity in broad range of pH 4.09.0 (Fig. S2).

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Fig. 2 SDS-PAGE analysis of the puried recombinant Llbglu1. Lane 1, puried recombinant Llbglu1, Lane 2 protein marker, broad range (7175 kDa)

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 nd the glycone specicity, kinetic constants like Km and Vmax were determined for these substrates using 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 puried recombinant Llbglu1 was incubated with amygdalin (Cyanogenic b-glucoside) and various avonoid glycosides such as genistein 7-O-glycosides, genistein 40 O-glycoside, apigenin 7-O-glycoside, naringenin 7-O-glycoside and kaempferol 3-O-glycoside as substrates, we found that Llbglu1 exhibited glycosidase activity towards amygdalin and avonoid glycosides except kaempferol 3-O-glycosides. The reaction product of amygdalin was analyzed 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]?) respectively. Flavonoid glycoside produces apigenin (avones) from apigenin 7-O-glycoside, genistein (isoavones) from genistein 7-O-glycosides and genistein 40 -O-glycoside, and naringenin (avanone) 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 qualitative information that Llbglu1 can hydrolyse natural substrates of hydroxynitrile glycosides and avonoid/ isoavonoid glycosides.

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

Homology modeling and active site identication 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 Rauvola serpentina, 49 % with Dhurrinase (1V02:E) from

When different substrates such as various nitro-phenylderived chromogenic substrates were tested for linkage specicity (Table 1) it is found that the enzyme can

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Fig. 4 HPLC chromatogram of Llbglu1 assay mixture with A Genistein 7-O-glucoside and B genistein 40 -O-glucoside, C Std1: genistein, D naringenin 7-O-glucoside, E Std2: naringenin, F apigenin

7-O-glucoside, G Std3: apigenin, H LCMS: positive ion mass spectrum of amygdalin. P1, P2 and P3 are genistein, naringenin and apigenin respectively

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 exible 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-glucosidase belonging to clan GH-A. The geometry of the nal rened model was evaluated with Ramachandrans plot (Fig. S3). From the pairwise structural alignment, it is quite evident 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 biological 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 hydrolysis mechanism. b-glucosidase of Zea 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) (corresponding 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).

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Mol Biol Rep Fig. 5 A Modeled Llbglu1: a-helices, b-sheets and loops are shown in red, yellow and green color 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 gure online)

Molecular docking of rhodiocyanoside A and other classes of glucosides Phylogenetic analysis of Leucaena b-glucosidase (Llbglu1) shows that it is closely related to Lotus japonicus b-glucosidases which hydrolyze the rhodiocyanoside A preferentially [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 nd its probable natural substrate. Docking of the rhodiocyanoside A in the active site of Llbglu1 (Fig. 5D) showed that it binds to the enzyme pocket with high afnity. 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 different 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 and 4.2 A from the Glu 199 tance of O2 were found 3.0 A 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 vicinity of the docked ligand 17 residues were found in 5 A 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). Denitely these aromatic rings containing amino acid have important

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Fig. 6 Interactions of catalytic residues of Llbglu1 with Rhodiocyanoside A through ligand exible t docking; A Ligplot: schematic diagrams of Llbglu1-Rhodiocyanoside A (proteinligand) interactions. 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 , Aglucon nitrogen in Rhodiocyanoside A Glu-413 are 3.0 and 4.2 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 representation) positioned in the binding cleft. D Locations of all the 17 residues (stick representation) forming the binding pocket containing docked Rhodiocyanoside A ligand. (Color gure online)

role to attract and hold the substrate till the end of the hydrolysis reaction. Tryptophan residue (Trp 385) of Llbglu1 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].

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Fig. 7 Docked complexes of modeled Llbglu1 with various glucosides. A Amygdalin, B rhodiocyanoside A, C genistein 7-O-glucoside, 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

All the other glycosides that were hydrolysed by Llbglu1, 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 rst choice of the substrates for the enzyme may belong to a class of hydroxynitrile glucosides. However, avonoid/isoavonoid 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 t 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 Glycosyl hydrolase family 1 b-glucosidase. Phylogenetic analysis shows that this b-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 properties of L. leucocephala b-glucosidase are in broad agreement with other plant b-glucosidases. The enzyme readily hydrolyzed a hydroxynitrile glycoside, amygdalin. Further, it also shows hydrolyzing activity towards avonoid/isoavonoid 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 identication of its natural substrate in vivo and may nd 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 Scientic and Industrial Research, New Delhi and to Anurag Misra by University Grants Commission, New Delhi is gratefully acknowledged.

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