196 Protein & Peptide Letters, 2009, 16, 196-200 0929-8665/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Trypanothione Reductase from Leishmania infantum: Cloning, Expres- sion, Purification, Crystallization and Preliminary X-Ray Data Analysis Paola Baiocco, Stefano Franceschini, Andrea Ilari and Gianni Colotti* Istituto di Biologia e Patologia Molecolari, CNR, and Dipartimento di Scienze Biochimiche, Università “Sapienza” di Roma, P.le A. Moro 5, I-00185 Rome, Italy Abstract: The most promising targets for Leishmania-specific drug design are two key enzymes involved in the unique thiol-based metabolism, common to all parasites of the Trypanosomatidae family: trypanothione synthetase (TryS) and trypanothione reductase (TR). Recently, new inhibitors of TR have been identified such as polyamines and tricyclic com- pounds. The knowledge of the three-dimensional structure of Leishmania TR will shed light on the mechanism of interac- tion of these inhibitors with TR and will be the starting point to design novel lead candidates to facilitate the development of new effective and affordable drugs. Trypanothione reductase from Leishmania infantum has been cloned, expressed in E. coli and purified. Crystals were obtained at 294 K by the hanging drop vapour diffusion method using ammonium sul- fate as precipitant agent and diffract to better than 2.95 Å resolution using a synchrotron radiation source. The crystals ex- hibit an unusually high solvent content of 74 %, belong to the tetragonal space group P41 with units cell parameters a=b=103.45 Å, c=192.62 Å and two molecules in the asymmetric unit. The protein X-ray structure has been solved by Molecular Replacement and the model is under construction. Keywords: Trypanothione reductase, Leishmania infantum, drug target, X-ray diffraction, molecular replacement. 1. INTRODUCTION Leishmania species are human parasites of the trypano- somatidae family causing severe illnesses generically known as visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). These diseases, spreading in India, Africa and South America, are strictly related to poverty and are characterised by high morbidity and mortality [1]. No vaccination is avail- able against leishmaniasis and fast spreading drug resistance is posing a major medical threat [2]. Trypanosoma and Leishmania differ from all other eukaryotes and prokaryotes in their specific redox metabolism, since they do not rely on the glutathione/glutathione reductase system to keep a stable redox balance which is based on the thiol–polyamine conju- gate trypanothione [3]. Trypanothione is essential in the parasite redox metabolism and is absent in mammalian cells. For this reason, the enzymes involved in the trypanothione metabolism are the most favourite candidates for the design of highly selective anti-parasitic drugs against Leishmaniasis [4, 5]. The enzyme involved in trypanothione biosynthesis is the trypanothione synthetase, which catalyzes the conjuga- tion of the polyamine spermidine and the tripeptide glu- tathione, and has no analogue in mammals. Trypanothione is kept reduced by the trypanothione reductase, which is similar (40% sequence identity) to human glutathione reductase (GR) but is highly specific to trypanothione and does not recognize glutathione as a substrate [6-11]. Trypanothione reductase is the most thoroughly studied enzyme of the try- panothione redox metabolism. Several reverse genetic ap- *Address correspondence to this author at the IBPM, CNR c/o Dept. Bio- chemical Sciences, University “Sapienza” of Rome, P.le A. Moro5, I-00185, Roma, Italy; Tel: +390649910910; Fax: +39064400062; E-Mail: gianni.colotti@uniroma1.it proaches have demonstrated that TR is essential in different Leishmania species [12, 13]. TR is a dimeric flavoprotein which uses NADPH as co-substrate to catalyze the reduction of trypanothione disulphide bonds. The current treatment of leishmaniasis relied mainly on arsenical and antimonial drugs, meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). They are potent inhibitors, interacting directly with the cys- teines in the active site of TR upon reduction to SbIII, and are supposed to compromise the thiol redox potential of the cell by inducing the efflux of intracellular thiols and by in- hibiting trypanothione reductase [14, 15]. However, an ever increasing number of drug-resistant strains have been identi- fied, which prevent the use of these drugs in the endemic areas and these drugs cause severe side effects [16-18]. Thus, there is an urgent need to develop novel drugs that target specific metabolic pathways of the parasite and the recent sequencing of the genomes of three Leishmania species, L. major, L. infantum, L. brasiliensis, opens unprecedented opportunities to tackle leshmaniasis and to develop new drugs for innovative treatment strategies. Recently, for these purpose new inhibitors of TR have been identified such as polyamines and tricyclic compounds that display low mi- cromolar inhibition constants [4, 5, 19-21]. The knowledge of the three-dimensional structure of Trypanothione reduc- tase of Leishmania infantum will shed light on the mecha- nism of interaction of these new inhibitors with TR and will be the starting point to design novel lead candidates to facili- tate the development of new effective and affordable drugs. With this purpose, trypanothione reductase from Leishmania infantum has been cloned, expressed, purified and crystal- lized and complete datasets on diffraction quality crystals have been collected at 2.95 Å resolution.