<p> Supplementary information</p><p>Functional characterization, homology modeling and docking studies of β- glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (Subabul)</p><p>Noor M. Shaika, Anurag Misrab, Somesh Singha, Amol B. Fatangarea, Suryanarayanarao</p><p>Ramakumarb, Shuban K. Rawala, Bashir M. Khana* aPlant Tissue Culture Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune -</p><p>411008, India bBioinformatics Centre, Department of Physics, Indian Institute of Science, Bangalore - 560012,</p><p>India</p><p>*Corresponding author: Dr. Bashir M. Khan, Senior Scientist and Head, Plant Tissue Culture Division, National</p><p>Chemical Laboratory, Dr. Homi Bhabha Road, Pune – 411008, India. </p><p>Tel.: +91-20-25902220, Fax: +91-20-25902645, E-mail: [email protected] Fig. S1 Multiple sequence alignment (MSA) of amino acid sequences of selected Glycosyl Hydrolase Family 1 β-glucosidases involved in defense along with L. leucocephala β- glucosidase (Llbglu1).</p><p>Fig. S2 Effect of pH on the enzymel activity of Llbglu1. The pH stability of the recombinant enzyme was determined by measuring the remaining activity (Residual Activity) under standard conditions (pH 4.8, 45 oC) after incubating the purified enzyme at 37 oC for different time intervals in buffers from pH 2 to 12. The following buffers (50 mM) were used for these studies: Glycine-HCl for pH 1.0-3.0, acetate buffer for pH 4.0-5.0, phosphate buffer for pH 6.0-7.0, Tris- HCl for pH 8.0-9.0 and Glycine-NaOH for pH 10.0-12.0. The residual activity is expressed here as a percentage of the maximum activity that is observed.</p><p>Fig. S3 Ramachandran (φ, ψ) plot for modeled Leucaena β-glucosidase (Llbglu1)</p><p>Fig. S4 Pair wise sequence alignment of Llbglu1 and 1CBG and their secondary structures. Coils and arrows represent helices and strands respectively. α, β, η and TT correspond to α- helix, β-strand, 3 -helix and β-turn respectively. 10</p><p>Fig. S5 Multiple structural alignments in sequence form. Sinapis alba myrosinase (1MYR:A), Sinapis alba myrosinase (1E4M:M), Sorghum bicolor dhurrinase1 (1V03:A), Sorghum bicolor dhurrinase1 (1V02:E), Sorghum bicolor dhurrinase1 (1V02:A), Basidiomycete β-glucosidase bgl1a ( 2E40:A), Maize β-glucosidase zmglu1 (1E56:A), white clover Trifolium repens β-glucosidase (1CBG:A), human cytosolic β- glucosidase (2JFE:X), Aphid myrosinase (1WCG:A), Leucaena leucocephala β- glucosidase (8LBG). Catalytic glutamic acid residues in conserved regions (ITLNEP/ITENG) a r e marked with arrow and Conserved residues involved in glycone binding pocket are shown in red. </p>