Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties.

Although ADP-dependent sugar kinases were first described in archaea, at present, the presence of an ADP-dependent glucokinase (ADP-GK) in mammals is well documented. This enzyme is mainly expressed in hematopoietic lineages and tumor tissues, although its role has remained elusive. Here, we report a detailed kinetic characterization of the human ADP-dependent glucokinase (hADP-GK), addressing the influence of a putative signal peptide for endoplasmic reticulum (ER) destination by characterizing a truncated form.

Structure and inhibition of SARS-CoV-1 and SARS-CoV-2 main proteases by oral antiviral compound AG7404.

Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (M), which presents structural similarity with the active site domain of enterovirus 3C protease, is one of the best-characterized drug targets of these viruses. Here we studied the antiviral activity of the orally bioavailable enterovirus protease inhibitor AG7404 against SARS-CoV-1 and SARS-CoV-2 from a structural, biochemical, and cellular perspective, comparing it with the related molecule rupintrivir (AG7800).

Free radicals derived from γ-radiolysis of water and AAPH thermolysis mediate oxidative crosslinking of eGFP involving Tyr-Tyr and Tyr-Cys bonds: the fluorescence of the protein is conserved only towards peroxyl radicals.

The enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis.

Protein topology determines substrate-binding mechanism in homologous enzymes.

During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders , , and , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts.

Bifunctional ADP-dependent phosphofructokinase/glucokinase activity in the order Methanococcales--biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis.

In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis.

Crystal structure, SAXS and kinetic mechanism of hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis reveal a conserved mechanism for catalysis.

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Thermococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function.