RESUMO
The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP(-) and was activated by free Mg(2+). On the other hand, HXE mutants did not affect the KM for MgADP(-), were still inhibited by free Mg(2+), and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP-AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.
Assuntos
Proteínas Arqueais/química , Glucoquinase/química , Glucose/metabolismo , Thermococcus/enzimologia , Motivos de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Sequência Conservada , Glucoquinase/genética , Glucoquinase/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Thermococcus/genéticaRESUMO
The tellurium oxyanion tellurite is harmful for most microorganisms. Since its toxicity occurs chiefly once the toxicant reaches the intracellular compartment, unveiling the toxicant uptake process is crucial for understanding the whole phenomenon of tellurium toxicity. While the PitA phosphate transporter is thought to be one of the main paths responsible for toxicant entry into Escherichia coli, genetic and physiological evidence have identified the ActP acetate carrier as the main tellurite importer in Rhodobacter capsulatus. In this work, new background on the role of these transporters in tellurite uptake by E. coli is presented. It was found that, similar to what occurs in R. capsulatus, ActP is able to mediate toxicant entry to this bacterium. Lower reactive oxygen species levels were observed in E. coli lacking the actP gene. Antioxidant enzyme catalase and fumarase C activity was almost unchanged after short exposure of E. coli ΔactP to sublethal tellurite concentrations, suggesting a low antioxidant response. In this strain, tellurite uptake decreased significantly during the first 5 min of exposure and inductively coupled plasma optical emission spectroscopy assays using an actP-overexpressing strain confirmed that this carrier mediates toxicant uptake. Relative gene expression experiments by qPCR showed that actP expression is enhanced at short times of tellurite exposure, while pitA and pitB genes are induced later. Summarizing, the results show that ActP is involved in tellurite entry to E. coli and that its participation occurs mainly at early stages of toxicant exposure.