Rafael Molina

Los efluentes procedentes de la parbolización del arroz contienen compuestos no biodegradables que resultan tóxicos para los ecosistemas acuáticos. En este estudio, se utilizó un catalizador de Fe altamente disperso sobre sílice obtenida de cascarilla de arroz (5Fe@SiO2) para la reacción de oxidación del fenol presente en aguas residuales de la parbolización del arroz. La reacción se hizo a 25 °C y a presión atmosférica. El catalizador alcanzó una reducción de la demanda química de oxígeno (DQO) del 67±3 %, una mineralización del 68,4±2,2 % y una conversión de fenol del 81,4±2 % después de 36 horas. El rendimiento del catalizador en la oxidación de fenol demuestra el potencial de aplicación del material en el tratamiento de aguas residuales. Dichos sistemas constituyen una estrategia para la conservación de los recursos naturales y la preservación de la biodiversidad en Colombia. Este estudio constituye una contribución a la estrategia de transición hacia la economía circular.

Background: Three out of the four classes of Porifera build siliceous skeletons, but they do this through divergent enzymatic pathways that are still poorly characterised. In demosponges, where the process is best known, the biosilicification occurs by polycondensation of silica using silicatein and other proteins (silica depolymerisers and scaffolding proteins) on an axis made of actin. For silicic acid intake, sponges use several types of membrane transporters with phylogenetic affinities to those in bacteria and plants. Most demosponges can produce more than one spicule type, sometimes beautifully ornamented, but this diversity is not clearly linked to the expression of polycondensing, scaffolding or transporter proteins. Here, we explore the evolution of the molecular toolkit for biosilicification in demosponges and across Porifera, using a time-calibrated phylogeny with an unparalleled dataset of 64 sponge genomes and transcriptomes. Results: Our results indicate a significantly more complex toolkit of genes involved in the biosilicification process than previously described, with a wide array of clade-specific proteins, especially silicateins, which allows us to hypothesise a link between silicatein type and diversity of spicules. We also describe specific putative expansions of the molecular complements for water and silicic acid transporters (aquaporins), some being acquired through horizontal gene transfer, potentially linked to adaptations to freshwater conditions. Conclusions: Our overview of the evolution of the biosilification process, with the most complete dataset to date, corroborates the hypothesis of independent origins for the main silicifying enzymes and provides a scenario of duplication and loss of genes that have allowed the massive diversification and evolutionary success of demosponges in a wide range of habitats.

Iron-sulfur clusters are essential cofactors for the accurate cellular function of many proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the action of the Cys desulfurase supercomplex, which is activated by the protein frataxin (FXN). The decrease of FXN expression and/or function results in Friedreich’s ataxia (FRDA). In this work, several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity and a strong interaction with FXN. Nanobody interaction stabilized wild-type and FRDA-related FXN variants in vitro. FXN-nanobody complexes were characterized by NMR, SAXS, and X-ray crystallography. Additionally, Nanobody expression was studied in human cells. The subcellular localization, direct interaction with FXN by in situ proximity ligation assay, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. Significantly, nanobody expression did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway. As a whole, our results suggest that nanobodies can serve as binding partners for mitochondrial FXN. However, the specific effect of the nanobodies on the conformational stability of FRDA-related FXN variants in cells should be investigated.

Lytic transglycosylases (LTs) belong to a family of enzymes that turnover the bacterial cell-wall peptidoglycan through a nonhydrolytic cleavage of the β(1-4) glycosidic bond, generating a hallmark 1,6-anhydromuramyl moiety in the reaction products. LTs are essential for numerous cellular processes, including cell-wall maturation, peptidoglycan recycling, cell division, and the assembly of multiprotein complexes. Their functional diversity underscores their biological significance. Family 3 LTs are distinguished by their EF-hand Ca²⁺-binding motif and are classified into two subfamilies. Subfamily 3B members, including Pseudomonas aeruginosa SltB2, possess a peptidoglycan-binding domain absent in subfamily 3A. In this study, we present the structural characterization of P. aeruginosa SltB2. The high-resolution crystal structure of SltB2 reveals a unique modular architecture shaped by the specific arrangement of its PG-binding domain and distinct differences in the organization of key residues surrounding the catalytic Glu residue compared to other family 3 members. A model of interaction between SltB2 and the peptidoglycan is proposed, which accounts for the enzyme's tolerance to peptide stems and reveals particular features at site +2, due to the unique arrangement of the PG-binding domain, explaining its preferred exolytic activity. Comparative structural analyses of Family 3 LTs provide insights into substrate recognition and enzymatic function, advancing our understanding of bacterial cell-wall remodeling mechanisms.

Spore germination in Clostridioides difficile is initiated by a cascade of activities of several proteins that culminates in the activation of SleC, a cell-wall-processing enzyme. We report herein the details of the enzymatic activities of SleC by the use of synthetic peptidoglycan fragments and of spore sacculi. The reactions include the formation of 1,6-anhydromuramate─a hallmark of lytic transglycosylase activity─as well as a muramate hydrolytic product, both of which proceed through the same transient oxocarbenium species. Furthermore, we report the first X-ray structure of zymogenic prepro-SleC at 2.1 Å resolution. Additionally, the structure provides insights into the YabG and CspB cleavage sites necessary for the activation of the zymogen. The active site of SleC presents relevant differences in contrast to SpoIID, a homologous lytic transglycosylase involved in the sporulation Clostridioides species, explaining the ability of SleC to turn over the spore sacculus, a prerequisite for the germination event. A screening of an in-house library of compounds led to the discovery of an oxadiazole that binds to the mature (activated) form of SleC, whereby it shuts down the ability of spores to germinate in the presence of germinants. This is consistent with the SleC activity as an end-point for the germination cascade. The mechanistic knowledge and the inhibitor hold the promise in addressing an unmet medical need in intervention of recurrent infections by C. difficile.

Bacteria and their phage adversaries are engaged in an ongoing arms race, resulting in the development of a broad antiphage arsenal and corresponding viral countermeasures. In recent years, the identification and utilization of CRISPR–Cas systems have driven a renewed interest in discovering and characterizing antiphage mechanisms, revealing a richer diversity than initially anticipated. Currently, these defense systems can be categorized based on the bacteria’s strategy associated with the infection cycle stage. Thus, bacterial defense systems can degrade the invading genetic material, trigger an abortive infection, or inhibit genome replication. Understanding the molecular mechanisms of processes related to bacterial immunity has significant implications for phage-based therapies and the development of new biotechnological tools. This review aims to comprehensively cover these processes, with a focus on the most recent discoveries.

Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA₄ cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.

Structure predictions have matched the accuracy of experimental structures from close homologues, providing suitable models for molecular replacement phasing. Even in predictions that present large differences due to the relative movement of domains or poorly predicted areas, very accurate regions tend to be present. These are suitable for successful fragment-based phasing as implemented in ARCIMBOLDO. The particularities of predicted models are inherently addressed in the new predicted_model mode, rendering preliminary treatment superfluous but also harmless. B-value conversion from predicted LDDT or error estimates, the removal of unstructured polypeptide, hierarchical decomposition of structural units from domains to local folds and systematically probing the model against the experimental data will ensure the optimal use of the model in phasing. Concomitantly, the exhaustive use of models and stereochemistry in phasing, refinement and validation raises the concern of crystallographic model bias and the need to critically establish the information contributed by the experiment. Therefore, in its predicted_model mode ARCIMBOLDO_SHREDDER will first determine whether the input model already constitutes a solution or provides a straightforward solution with Phaser. If not, extracted fragments will be located. If the landscape of solutions reveals numerous, clearly discriminated and consistent probes or if the input model already constitutes a solution, model-free verification will be activated. Expansions with SHELXE will omit the partial solution seeding phases and all traces outside their respective masks will be combined in ALIXE, as far as consistent. This procedure completely eliminates the molecular replacement search model in favour of the inferences derived from this model. In the case of fragments, an incorrect starting hypothesis impedes expansion. The predicted_model mode has been tested in different scenarios.

The essential membrane complex FtsE/FtsX (FtsEX), belonging to the ABC transporter superfamily and widespread among bacteria, plays a relevant function in some crucial cell wall remodeling processes such as cell division, elongation, or sporulation. FtsEX plays a double role by recruiting proteins to the divisome apparatus and by regulating lytic activity of the cell wall hydrolases required for daughter cell separation. Interestingly, FtsEX does not act as a transporter but uses the ATPase activity of FtsE to mechanically transmit a signal from the cytosol, through the membrane, to the periplasm that activates the attached hydrolases. While the complete molecular details of such mechanism are not yet known, evidence has been recently reported that clarify essential aspects of this complex system. In this chapter we will present recent structural advances on this topic. The three-dimensional structure of FtsE, FtsX, and some of the lytic enzymes or their cognate regulators revealed an unexpected scenario in which a delicate set of intermolecular interactions, conserved among different bacterial genera, could be at the core of this regulatory mechanism providing exquisite control in both space and time of this central process to assist bacterial survival. Keywords Bacterial division Divisome FtsE FtsX PcsB Lytic regulation SPOR 3D structure Mechano-transmission ABC transporters

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA₄ rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA₄ phosphodiester hydrolysis in a non-concerted manner.