César A. Ramírez-Sarmiento

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Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and(More)
Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single-molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic adenine diphosphate (ADP)-dependent glucokinase from(More)
Pyridoxal 5′-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we(More)
Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a(More)
Most proteins, in order to perform their biological function, have to fold to a compact native state. The increasing number of knotted and slipknotted proteins identified suggests that proteins are able to manoeuvre around topological barriers during folding. In the present article, we review the current progress in elucidating the knotting process in(More)
The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na(+). Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to(More)
RfaH is a virulence factor from Escherichia coli whose C-terminal domain (CTD) undergoes a dramatic α-to-β conformational transformation. The CTD in its α-helical fold is stabilized by interactions with the N-terminal domain (NTD), masking an RNA polymerase binding site until a specific recruitment site is encountered. Domain dissociation is triggered upon(More)
The forkhead family of transcription factors (Fox) controls gene transcription during key processes such as regulation of metabolism, embryogenesis, and immunity. Structurally, Fox proteins feature a conserved DNA-binding domain known as forkhead. Interestingly, solved forkhead structures of members from the P subfamily (FoxP) show that they can oligomerize(More)
Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then(More)
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(More)