626 Current Pharmaceutical Biotechnology, 2009, 10, 626-630 1389-2010/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. In Silico Studies on Tryparedoxin Peroxidase of Leishmania infantum: Structural Aspects Bishal Kumar Singh and Vikash Kumar Dubey* Department of Biotechnology, Indian Institute of Technology, Guwahati-781039, Assam, India Abstract: Tryparedoxin peroxidase (TryP) is a key enzyme of the trypanothione-dependent metabolism for removal of oxidative stress in leishmania. These enzymes function as antioxidants through their peroxidase and peroxynitrite reduc- tase activities. Inhibitors of this enzyme are presumed to be antilesihmania drugs and structural studies are prerequisite of rational drug design. We have constructed three dimensional structure of TryP of Leishmania infantum using comparative modeling. Structural analysis reveals several interesting features. Moreover, it shows remarkable structural difference with human host glutathione peroxidase, an enzyme involved in similar function and TryP from Leishmania major. Keywords: Drug design, Structure-Function, Peroxidase activity, Homology Modelling. INTRODUCTION Every organism living in an aerobic environment expose to reactive oxygen species (ROS) such as the superoxide anion (O 2 - ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH . ) which are potentially destructive, capable of oxidizing proteins or lipids and causing modification in nu- cleic acids [1]. In mammalian cells, the principle route of ROS detoxification involves superoxide dismutase and a variety of peroxidases including selenium dependent glu- tathione peroxidase working in concert with glutathione, glutathione reductase and NADPH [2]. However, parasitic lishmania, of order Kinetoplastida, lack this antioxidant de- fense mechanism and instead rely on a unique but analogous system involving cascade of three enzymes: trypanothione synthetase (EC 6.3.1.9), trypanothione-recycling flavopro- tein trypanothione reductase (EC 1.8.1.12), and trypare- doxin-recycling enzyme tryparedoxin peroxidase (TryP) working in concert with trypanothione (N 1 , N 8 -bis(gluta- thionyl)-spermidine), tryparedoxin (TryX), and NADPH [3- 5]. Thus, TryP is an attractive target enzyme for anti- leishmania drug development studies. TryP of Leishmania infantum, a 196 amino-acid enzyme with a molecular mass of approximately 21.5 kDa, is a member of typical 2-Cys peroxiredoxin super family which consists both peroxidase and peroxynitrite reductase activity [6]. TryP from Leishmania species. is reported to be mono- meric tryparedoxin-dependent peroxidases distinct from TryP of other Trypanosoma which are oligomer like decamer in Crithidia facsiculata and dimer in Trypanosoma cruzi. [7, 8]. TryP uses reduced tryparedoxin as an electron donor and catalyze the reduction of hydrogen peroxide and organic hydro-peroxides to water or alcohol [8]. Tryparedoxin is reduced by dihydrotrypanothione, which in turn maintained in high level by flavoprotein trypanothione reductase at the expense of NADPH. Thus, in Leishmania infantum, as in all *Address correspondence to this author at the Department of Biotechnology, Indian Institute of Technology, Guwahati-781039, Assam, India; Tel: 91- 361-2582203; Fax: 91-361-2582249; Email: vdubey@iitg.ernet.in Kinetoplastida, tryparedoxin peroxidase is the final electron acceptor of a unique trypanothione system for detoxifying hydroperoxides, constituting a relevant target for develop- ment of new drugs by rational inhibitor design. RESULT AND DISCUSION Tryparedoxin peroxidase from Trypanosoma cruzi (PDB accession code 1UUL) was found as most identical sequence having lowest E-value 6e-10 and 69% sequence identity with target TryP of Leishmania infantum. However the next clos- est was tryparedoxin peroxidase from Crithidia fasciculata (PDB accession code 1E2Y) which shows E-value 2e-80 and 72% sequence identity with target enzyme. Thus, they have been used as templates for homology modelling purpose. Siquence alignment of TryP enzymes from Crithidia fascicu- lata, Trypanosoma cruzi and target Leishmania infantum is shown in Fig. (1). Out of 196 residues 115 were conserved across these sequences that will be discussed later in the con- text of model structure. The geometry of the main chain and side chains have been analyzed using the program PROCHECK [9]. Ramachandran plot [10] for the derived model of TryP of Leishmania infantum depicted in Fig. (2) shows that the and backbone torsion angles of most of the non-glycine residues are within the allowed regions of which 95.3% are in the energetically most favored area. Only 4.7% residues are found in additional allowed regions. Be- sides, no residues are in disallowed regions. The modelled TryP from Leishmania infantum has 0.42 and 0.46 Å RMS deviation to all C atoms with template structure, TryP from Crithidia fasciculate and Trypanosoma cruzi, respectively which is acceptable. The structural comparison of modeled TryP of Leishma- nia Infantum with that of Crithidia fasciculate and Trypano- soma cruzi shows that the secondary structure and domain organization are fairly well conserved. The secondary struc- ture analysis was done using Swiss PDB viewer [11] which is mapped on the amino acid sequence in Fig. (1). The over- all structure of TryP of Leishmania Infantum has approxi- mately 26% of the residues in six alpha-helices of different