Trypanothione reductase from Leishmania infantum: cloning, expression, purification, crystallization and preliminary X-ray data analysis.
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 compounds. The knowledge of the three-dimensional structure of Leishmania TR will shed light on the mechanism of interaction 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 sulfate as precipitant agent and diffract to better than 2.95 A resolution using a synchrotron radiation source. The crystals exhibit an unusually high solvent content of 74 %, belong to the tetragonal space group P41 with units cell parameters a=b=103.45 A, c=192.62 A and two molecules in the asymmetric unit. The protein X-ray structure has been solved by Molecular Replacement and the model is under construction.
Baiocco
Paola
P
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. gianni.colotti@uniroma1.it.
Franceschini
Stefano
S
Ilari
Andrea
A
Colotti
Gianni
G
D000818:Animals; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018314:Leishmania infantum / Q000201:enzymology* / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase