RESUMEN
Statistics from structural genomics initiatives reveal that around 50-55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748T produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-ß-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer.
Asunto(s)
Glicósido Hidrolasas/química , Lactobacillus plantarum/química , Soluciones/química , Dominio Catalítico , Cisteína/química , Cisteína/metabolismo , Glucosidasas/química , Glucosidasas/metabolismo , Glicósido Hidrolasas/metabolismo , Lactobacillus plantarum/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Proteolisis , Serina/química , Serina/metabolismo , Solubilidad , Especificidad por SustratoRESUMEN
Chagas disease, also known as American trypanosomiasis, is caused by infection with the protozoan parasite Trypanosoma cruzi. The Pan American Health Organization (PAHO) estimates that currently 7.7 million of people have Trypanosoma cruzi infection in the 21 endemic countries from the southern and southwestern United States to central Argentina and Chile. The only approved therapeutics for the treatment of Chagas disease are two nitroheterocyclic compounds as a nitrofuran (nifurtimox; Lampit) and a nitroimidazole (benznidazole; Rochagan). However, the anti-Trypanosoma cruzi activities of these compounds were discovered empirically over three decades ago. The treatment of Chagas disease with nifurtimox or benznidazole is unsatisfactory because of their limited efficacy in the prevalent chronic stage of the disease and their toxic side effects. In this context, this article will review the current knowledge of the different aspects involved in this illness, such as Trypanosoma cruzi transmission, physiology and biochemistry of the etiological agent, epidemiological aspects and current treatments for American trypanosomiasis. An important section of this review will focus on the different strategies in drug discovery for Chagas disease, including methodology, in vitro screening studies against whole parasites, novel rationally developed approaches on the basis of the increasing knowledge of the biochemistry of Trypanosoma cruzi and the recent progress in the understanding and validation of several targets for the therapy of Chagas's disease. A summary of the most relevant drug targets such as sterol biosynthesis pathway, cysteine protease pathway, pyrophosphate metabolism and purine salvage pathway will be reviewed. Moreover, recent studies regarding other strategies currently under development including thiol-dependent redox metabolism, lysophospholipid analogues and DNA binders will also be discussed.