RESUMO
Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221 , with unit-cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.
Assuntos
Fumarato Hidratase/química , Fumarato Hidratase/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Fumarato Hidratase/genética , HEPES/química , HEPES/metabolismo , Humanos , Cinética , Lisina/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86-Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs.