IN SILICO ANALYSIS OF INHIBITOR AND SUBSTRATE BINDING SITE OF SERRAPEPTIDASE FROM SERRATIA MARCESCENS MTCC 8708 1N.S.Kaviyarasi1, C.N.Prashantha 2, V.V.S.Suryanarayana3 School of Natural Science, Bangalore university, Bangalore - 560 056, India, 2Division of Bioinformatics, Scientific Bio-Minds-560092  3Indian Veterinary Research Institute, Hebbal, Bangalore - 560 024, India BACK GROUND Figure 4: Contact map of Serrapeptidase with Substrates RESULTS & DISCUSSION Serrapeptidase is a therapeutic enzyme generally used as anti-inflammatory drug. It is a metalloprotease belonging to serralysin family. It was first isolated in late 1960s from Serratia E15 [1]. It is available as tablet or capsule form, Medindia's database currently has 1012 Brands of Generics of Serratiopeptidase listed [2]. Available in fixed dose combination with NSAIDs (Diclofenac sodium, Nimesulide, Paracetamol). NSAIDs have potent anti-inflammatory, analgesic and antipyretic actions. It inhibits the enzyme, cyclooxygenase, thus resulting in reduced synthesis of prostaglandin precursors [3]. Serrapeptidase a metalloprotease appears to have hydrolyzing activity of various peptides such as histamine, bradykinin, substance P, serotonin, etc., The objective of present study is in silco analysis of the active site for substrate binding and confirm the active site structure using an inhibitor, . The Serrapeptidase gene is amplified from genomic DNA of Serratia marcescens MTCC 8707 [4] . SRP template protein is used to build model of Serrapeptidase 3D structure. The docking studies shows substrates bradykinin and substance-P bind near zinc binding site with minimum RMSD value(Fig.3,4) and the inhibitors EDTA and lisinopril showed favourable interaction at zinc binding site of serrapeptidase with minimum free energy ( Fig 6, Table. 1) Inhibitor docking: b Figure 5: Structure of EDTA and Lisinopril a Figure 6: EDTA and Lisinopril binding with Serrapeptidase Figure 1: 3D model structure of Serrapeptidase showing, calcium binding loops (a) , Zinc binding amino acid residue (b). The C-terminal domain is a β strand-rich domain containing eighteen β strands, a short α-helix and seven Ca2+ ions bound to calcium binding loops (Fig. 1a), which are not essential for its catalytic function, but are required for protection from autolysis. N- terminal zinc-binding domain and zinc ion is bound by His 192, His 196, His 202, Tyr 232 and the water molecule is in a distorted trigonal bipyramidal manner because it belongs to Met-zincins- ‘zincins superfamily’ with topologies of M-turn (HExxHxxGxxH…xxM) (Fig.1b). METHODOLOGY Table 1: Clustering results obtained from docking of inhibitors with Serrapeptidase Gene used The Serrapeptidase gene was amplified from genomic DNA of Serratia marcescens MTCC 8707 Genbank ID The deduced amino acid sequence of Serrapeptidase gene was used in this study (GI: KP869847) Protein folding The three dimensional structures was predicted using Swiss Model workspace [5]. Substrate docked The CABS-dock [6] was used for SRP-peptide docking Bradykinin (RPPGFSPFR), Substance-P (RPKPQQFFGLM) were used as substrates. (Fig. 2) Inhibitors docked The Swiss-Dock server [7] was used for docking Serrapeptidase with inorganic inhibitors like EDTA and Lisinopril (Fig. 5). Visualization & analysis The results obtained from CABS-Dock and Swiss Dock were pictured by UCSF Chimera. SI.No. Ligands Receptor No. of Swiss Dock Clusters Cluster rank Full fitness (Kcal/mol) Cluster Estimated ΔG (Kcal/mol) Remarks 1. Ethylenediaminetetraacetic acid dipotassium salt dihydrate EDTA ZINC 19364242 SRP 30 (250) -1742.31 -11.19 Binds near Zn binding site 1 2 -1742.18 -11.18 3 -1742.11 -11.17 -1740.43 -10.70 -1735.25 -9.74 2. Lisnopril ZINC 3812863 -1830.91 -10.50 . -1830.66 -10.47 4 -1829.39 -10.39 5 Substrate docking: Figure 2: Sturcture of (a) Bradykinin and (b) Substance-P a b CONCLUSION The structural elucidation shows that Serrapeptidase belongs to Serralysin family member. The result of docking studies confirm that the substrate or inhibitor binds near zinc binding domain (HEXXH...) and the peptide bond of substrate can be effectively cleaved by serrapeptidase. Hence as like other metallo-endopeptidase, the catalytic mechanism hypothesized to be hydrolysis of internal alpha peptide bonds in a polypeptide chain by a mechanism in which water acts as a nucleophile, a metal ion hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions. Figure 3: Bradykinin and Substance-P binding with Serrapeptidase REFERENCE Kodama R, Nakasuji Y, Yamauchi S, Nishio,M: J. Sericult.Sci., Japan 34 ;1965: 206. 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