survival comes from studies where CYP450 inhibitors, the azole antifungals namely itraconazole, ketoconazole and fluconazole, have been used as successful antileishmanial agents [9]. Azole antifungals also cause radical parasitological cure in murine models of Chagas’ disease, caused by a related parasite, the Trypanosoma cruzi through inhibition of CYP51, a sterol C14 alpha- demethylase [9–10]. Reportedly, Leishmania cell lysates catalyse CYP450-like reactions [11]. The above studies clearly indicate a functional role of CYP450s in kinetoplastid parasite biology; however, no reports are available on the actual functional involvement of these molecules. Due to their involvement in multiple functional systems, CYP450s are potential drug targets as shown in Mycobacterium tuberculosis [12] and are reportedly associated with drug resistance in Candida albicans [13]. One of the important functions of CYP450s is biosynthesis of ergosterol [14] which is the primary component of the Leishmania membrane and is functionally linked to maintenance of structural integrity and protection from biotic stress [15]. Therefore, interference with ergosterol biosynthesis could result in disruption of parasite function and molecules involved in this pathway could serve as potential drug targets [16]. Interestingly, Leishmania can survive altered sterol levels [17] but changes in sterol profile have been linked to amphotericin B and fluconazole resistance [9,18]. The genome database of the kinetoplastid parasites annotate three putative CYP450-like proteins amongst the members of the genus Leishmania and Trypanosoma, however, functional information on none of the three CYP450-like proteins is available which could have important role in Leishmania survival related to drug response, synthesis of biologically important molecules and elimination of xenobiotics. Therefore, it is important to analyse the function of Leishmania CYP450s to understand multiple functional aspects of parasite physiology that contribute to cell survival and drug resistance. In this study, we identify CYP5122A1 as a novel CYP450 of Leishmania donovani playing a role in several important events in parasite function including response to drugs and the ability to infect. Results CYP5122A1 is well conserved within Leishmania spp. To probe into the function of one of the CYP450-like proteins in Leishmania donovani, the gene was cloned using primers designed from the sequence of its orthologue in L. major (Lmj F27.0090, Gene DB, Wellcome Trust Sanger Institute, Hinxton, UK). The sequence of the full length clone (Acc. No. DQ267494) was classified by the ‘Cytochrome P450 Nomenclature Committee’ [19] as CYP5122A1 (Nelson D.R. at The Cytochrome P450 Homepage, http://drnelson.uthsc.edu/CytochromeP450.html; personal communication). The CYP5122A1 sequence shared close identities with CYP450–like proteins of other members of the Leishmania spp. like Leishmania major, Leishmania infantum, Leishmania mexicana, Leishmania braziliensis (87–98%) as well as with the Trypanosoma spp. (50–53%). A phylogenetic tree comparing CYP5122A1 with CYP450s from other species show Leishmania CYP450s as a separate cluster from Trypanosoma which is the closest neighbour, the evolutionary distance from mammals being large (Fig. S1). A characteristic feature of P450 superfamily is a high conservation in their general topology and structural fold despite low sequence similarity; however, no two CYP450s are structurally identical [20]. The CYP5122A1 sequence showed the presence of a transmembrane domain (TM; amino acids 38–60), a proton transfer groove (PTC; amino acid 348–353), a motif for CYP5122A1, a Novel Cytochrome P450 of Leishmania stabilization of the core (SC; amino acid 418–421) and a heme binding loop (HBL; amino acid 516–525) (Fig. 1A) that are characteristics of CYP450 proteins. Homology modelling attempts for CYP5122A1 on the RCSB (Research Collaboratory for Structural bioinformatics) PDB (www.pdb.org/) server identified several templates of CYP102 (BM-3) usable for homology modelling of CYP5122A1. One such template 1ZOA generated a model of CYP5122A1 spanning from 70 to 590 of 592 residues including the active site heme binding motif containing the characteristic P450 consensus sequence represented by Phe-Ile- Asn-Gly-Pro-Arg-Asn-Cys-Leu-Gly (amino acids 516 to 525). The conserved catalytic cysteine (marked by arrow), a ligand for the heme iron, occupies position 523 (Fig. 1B, Fig. S2), in the heme binding loop (Fig. 1A), and is exposed in a pocket just before the L helix (Fig. 1B; Fig. S2), as is observed in all the cytochrome P450s in general. The conserved motif for stability of the CYP450s (SC; Fig. 1A) was represented by Glu-Thr-Leu-Arg (amino acids 418 to 421; Fig. S2) located in the helix K on the proximal side of the heme binding domain (Fig. 1B, Fig. S2). The proton transfer groove (PTG; Fig. 1A) containing amino acids Ala-Gly-His-Glu- Thr-Ser (amino acids 348 to 353; Fig. S2), located on the distal side of the heme in the central part of I-helix was close to the substrate binding site (Fig. 1B). The overall packing quality of the model assessed using WHATCHECK server was within normal range (z = 21.329). Further analysis of the Ramachandran plot of the same generated by PROCHECK, showed majority (85%) of amino acids in the highly favourable region (Fig. S3). Therefore, the above data shows that CYP5122A1 of L. donovani contains CYP450 consensus sequences and shares close identity with CYP450 proteins of both the old and the new world Leishmania spp. CYP5122A1 shows no major change during differentiation of metacyclics and is located at different subcellular locations Parasite growth and differentiation into infective forms (metacyclics) within the insect host can be mimicked in vitro by culturing flagellated promastigotes [21]. To check intracellular levels of CYP5122A1, a specific antibody raised against the most immunodominant sequence stretch on CYP5122A1 that recog- nized a single band in Leishmania donovani whole cell lysates was used. When the presence of CYP5122A1 in the promastigotes and the amastigotes was checked, both forms showed the presence of CYP5122A1, where the total concentration of the protein appeared to be higher in the amastigotes (Fig. 2A). As shown in Fig. 2B, no major change in the intracellular CYP5122A1 levels was observed during differentiation of the metacyclics with the constitutive levels remaining fairly constant in an unsynchronized culture (Fig. 2C, bar graph). However, when we looked at protein levels in metacyclic parasites from logarithmic (day 3) and stationary phase (day 5), a higher CYP5122A1 protein level was observed in the metacyclics isolated from stationary phase cultures (Fig. 2D, lane 2) in comparison to those isolated from log phase cultures (Fig. 2D, lane 1). CYP5122A1 was associated with the lipid fraction and not the aqueous fraction of cell extracts prepared with Triton-X114 (Fig. 3A) suggesting membrane association of the protein which is in concurrence with the presence of a membrane spanning domain between amino acids 38–60 (Fig. 1A). Promastigotes from log phase cultures stained with anti-CYP5122A1 and counter-stained with BIP, an hsp70 family protein found in ER, and ER Tracker BlueH, a dapoxyl dye that stains ER in live cells, showed co- localization of ER and CYP5122A1 stain indicating localization of the protein to the ER (Fig. 3B, a–e, BIP and ER Tracker blue). In PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25273