Cloning, characterization and preliminary crystallographic analysis of Leishmania hypoxanthine-guanine phosphoribosyltransferase.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae hgprt gene contains a 633-nucleotide open reading frame that encodes a 23.4-kDa protein. A pairwise alignment of the different HGPRT's sequences revealed a 26%-53% sequence identity with the Leishmania sequences and 87% identity to the HGPRT of Leishmania donovani. A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The steady-state kinetic parameters were determined for the recombinant enzyme and the enzyme is active as a homodimer in solution. Single crystals were obtained for the L. tarentolae HGPRT representing the first Leishmania HGPRT crystallized and initial crystallographic data were collected. The crystals obtained belong to the orthorhombic space group (P2(1)2(1)2(1)) with unit cell parameters a=58.104 A, b=85.443 A and c=87.598 A and diffract to a resolution of 2.3 A. The availability of the HGPRT enzyme from Leishmania and its crystallization suitable for X-ray diffraction data collection should provide the basis for a functional and structural analysis of this enzyme, which has been proposed as a potential target for rational drug design, in a Leishmania model system.
Monzani
Paulo S
PS
Laboratory of Protein Crystallography and Structural Biology, Physics Institute of São Carlos, University of São Paulo-USP, Av. Trabalhador Sãocarlense 400, São Carlos, Brazil.
Alfonzo
Juan D
JD
Simpson
Larry
L
Oliva
Glaucius
G
Thiemann
Otavio H
OH
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007891:Leishmania / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase