RESUMEN
Human L-asparaginase-like protein 1 (ASRGL1) has hydrolytic activity against L-asparagine and isoaspartyl dipeptides. As an N-terminal nucleophile hydrolase family member, its activation depends on an intramolecular autoprocessing step between G167 and T168. In vitro, autoprocessing reaches only 50% completion, which restrains the activity and hampers the full understanding of the activation process. The ASRGL1 dimer interface plays a critical role in intramolecular processing, and the interactions within oligomers can offer relevant information about autoprocessing. In this work, a fully processed trimeric conformation of ASRGL1 was observed for the first time, and we combined biophysical and structural proteomics assays to characterize trimeric ASRGL1. Our analyses show that oligomerization is critical for autoprocessing, hydrolytic activity and thermal stability. The newest trimeric ASRGL1 conformation enhances protein activity and presents a melting temperature deviation of 4.33 °C in comparison to the monomeric conformation. The interaction of the third monomer in the trimeric conformation is driven by an α-helix comprising residues KVNLARLTLF (227-236).
Asunto(s)
Asparaginasa , Autoantígenos , Humanos , HidrólisisRESUMEN
A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the ß-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall ß-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/ß-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by â¼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.
Asunto(s)
Bacillus subtilis/enzimología , Celulosa/metabolismo , Endo-1,3(4)-beta-Glucanasa/metabolismo , Lacasa/metabolismo , Ingeniería de Proteínas/métodos , Saccharum/metabolismo , Celulosa/química , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/genética , Cinética , Lacasa/química , Lacasa/genética , Simulación de Dinámica Molecular , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharum/químicaRESUMEN
The growth of cells is controlled by stimulatory or inhibitory factors. More than twenty different families of polypeptide growth factors have been structurally and functionally characterized. Basic fibroblast growth factor (bFGF) of the fibroblast growth factor family was characterized in 1974 as having proliferative activity for fibroblastic cells. The inhibitory effects of ethanol on cell proliferation result from interference with mitogenic growth factors (e.g., bFGF, EGF and PDGF). In order to better understand the mode of action of bFGF, particularly regarding the influence of ethanol on the biological activity of bFGF, three recombinant bFGF mutants were produced (M6B-bFGF, M1-bFGF and M1Q-bFGF). In the present study, wild bFGF and these mutants were examined by molecular dynamics simulations in systems consisting of a solute molecule in ethanol solution at 298 K and physiological pH over 4.0 ns. The hydrogen bonds, the root mean square deviations and specific radial distribution functions were employed to identify changes in the hydrogen bond structures, in the stability and in the approximation of groups in the different peptides to get some insight into the biological role of specific bFGF regions. The detailed description of the intramolecular hydrogen bonds, hydration, and intermolecular hydrogen bonds taking place in bFGF and its mutants in the presence of ethanol established that the residues belonging to the beta5 and beta9 strands, especially SER-73(beta5), TYR-112(beta9), THR-114(beta9), TYR-115(beta9), and SER-117(beta9), are the regions most affected by the presence of ethanol molecules in solution.
Asunto(s)
Simulación por Computador , Etanol/farmacología , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Enlace de Hidrógeno/efectos de los fármacos , Modelos Químicos , Modelos Genéticos , Estructura MolecularRESUMEN
The growth of cells is controlled by stimulatory or inhibitory factors. More than twenty different families of polypeptide growth factors have been structurally and functionally characterized. Basic fibroblast growth factor (bFGF) of the fibroblast growth factor family was characterized in 1974 as having proliferative activity for fibroblastic cells. The inhibitory effects of ethanol on cell proliferation result from interference with mitogenic growth factors (e.g., bFGF, EGF and PDGF). In order to better understand the mode of action of bFGF, particularly regarding the influence of ethanol on the biological activity of bFGF, three recombinant bFGF mutants were produced (M6B-bFGF, M1-bFGF and M1Q-bFGF). In the present study, wild bFGF and these mutants were examined by molecular dynamics simulations in systems consisting of a solute molecule in ethanol solution at 298 K and physiological pH over 4.0 ns. The hydrogen bonds, the root mean square deviations and specific radial distribution functions were employed to identify changes in the hydrogen bond structures, in the stability and in the approximation of groups in the different peptides to get some insight into the biological role of specific bFGF regions. The detailed description of the intramolecular hydrogen bonds, hydration, and intermolecular hydrogen bonds taking place in bFGF and its mutants in the presence of ethanol established that the residues belonging to the beta5 and beta9 strands, especially SER-73(beta5), TYR-112(beta9), THR-114(beta9), TYR-115(beta9), and SER-117(beta9), are the regions most affected by the presence of ethanol molecules in solution.