Thermodynamics, functional and structural characterization of inosine–uridine nucleoside hydrolase from Leishmania braziliensis
| Autor: | Guilherme Oliveira Petersen, Osmar Norberto de Souza, Fernanda Teixeira Subtil, Anne Drumond Villela, Luiz Augusto Basso, Pedro Ferrari Dalberto, Leonardo K. Martinelli, Adilio da Silva Dadda, Luiza Galina, José Fernando Ruggiero Bachega, Luis Fernando Saraiva Macedo Timmers, Pablo Machado, Edgar Marcelino de Carvalho Filho, Diógenes Santiago Santos, Kenia Pissinate, Cristiano Valim Bizarro, Antonio Pinto |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
biology General Chemical Engineering Cytidine General Chemistry biology.organism_classification Leishmania braziliensis Uridine 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Biochemistry chemistry Hydrolase medicine Inosine Purine metabolism Nucleotide salvage Nucleoside medicine.drug |
| Zdroj: | RSC Adv.. 7:48861-48875 |
| ISSN: | 2046-2069 |
| DOI: | 10.1039/c7ra07268f |
| Popis: | 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 (LbIU-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 LbIU-NH steady-state kinetic constants for inosine, adenosine, cytidine, uridine and p-nitrophenyl β-D-ribofuranoside is also reported. These data suggest that LbIU-NH displays characteristics of a nonspecific hydrolase. The thermodynamic profile suggests that D-ribose can bind to free enzyme with favorable enthalpic (ΔH) and entropic (ΔS) contributions. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the LbIU-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 LbIU-NH in complex with β-D-ribose and Ca2+ at 1.5 A resolution is described. |
| Databáze: | OpenAIRE |
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