Thermodynamics, functional and structural characterization of inosine–uridine nucleoside hydrolase from Leishmania braziliensis. Issue 77 (17th October 2017)

• Record Type: Journal Article
• Authors: Dalberto, Pedro Ferrari; Martinelli, Leonardo Kras Borges; Bachega, Jose Fernando Ruggiero; Timmers, Luis Fernando Saraiva Macedo; Pinto, Antonio Frederico Michel; Dadda, Adilio da Silva; Petersen, Guilherme Oliveira; Subtil, Fernanda Teixeira; Galina, Luiza; Villela, Anne Drumond; Pissinate, Kenia; Machado, Pablo; Bizarro, Cristiano Valim; de Souza, Osmar Norberto; de Carvalho Filho, Edgar Marcelino; Basso, Luiz Augusto; Santos, Diogenes Santiago
• Is Part Of: RSC advances. Volume 7:Issue 77(2017)
• Page Start: 48861
• Page End: 48875
• Publication Date: 2017-10-17
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c7ra07268f
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : Inosine–uridine nucleoside hydrolase from Leishmania braziliensis is a nonspecific enzyme that contains a disulfide bond not needed for tetramer stabilization. Abstract : Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries with the highest incidence of cutaneous and mucocutaneous forms of leishmaniasis caused mainly by Leishmania braziliensis . The first-line drugs used in the treatment of leishmaniasis have several limitations: parenteral administration, long duration of treatment, and serious toxicity. One key metabolic characteristic of these parasites is the lack of a de novo purine biosynthesis pathway, making them auxotrophic to purines. Accordingly, they rely solely on the purine salvage pathway for nucleotide synthesis. A better understanding of the purine salvage pathway can reveal details of the biology of L. braziliensis that could, in turn, be used to develop new strategies to combat this parasite. The inosine–uridine nucleoside hydrolase from L. braziliensis ( Lb IU-NH) plays an important role in the salvage process and is an attractive drug target as there is no similar catalytic activity in mammals. Here is described cloning, heterologous protein expression, and a three-step purification protocol that yielded homogenous recombinant protein. The determination of Lb IU-NH steady-state kinetic constants for inosine, adenosine, cytidine, uridine and p -nitrophenyl β-d -ribofuranoside is also reported. These data suggest that Lb IU-NH displays characteristics of a nonspecific hydrolase. The thermodynamic profile suggests thatd -ribose can bind to free enzyme with favorable enthalpic (Δ H ) and entropic (Δ S ) contributions. Thermodynamic activation parameters ( E a, Δ G #, Δ S #, Δ H # ) for the Lb IU-NH-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, and pH-rate profiles are also presented. In addition, the crystal structure of Lb IU-NH in complex with β-d -ribose and Ca 2+ at 1.5 Å resolution is described.