ITQB: MMPR - Artigos em revista internacional com arbitragem científica
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- Regulation of bacterial haem biosynthesisPublication . Zamarreño Beas, Jordi; Videira, Marco A.M.; Saraiva, Lígia M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ElsevierHaem b and sirohaem are two iron-chelated modified tetrapyrroles that serve as prosthetic groups in proteins with crucial roles in a variety of biological functions, such as gas transport, respiration, and nitrite and sulphite reduction. These tetrapyrroles are synthesised from 5-aminolaevulinic acid and share a common pathway until the formation of uroporphyrinogen III, from where the synthesis diverges. In bacteria, sirohaem is produced from uroporphyrinogen III through the activities of one, two or three separate proteins, while haem b is synthesised through three distinct pathways. The biosynthesis of haem b and sirohaem comprises intermediates and end-products that are unstable or potentially hazardous to the cell. Therefore, the cellular metabolic fluxes of tetrapyrroles need to be tightly controlled by substrate channelling and/or other regulatory processes. This review summarises the recent advances on the regulation and protein–protein interactions controlling the formation of sirohaem and haem b in bacteria.
- Metabolomics of Escherichia coli Treated with the Antimicrobial Carbon Monoxide-Releasing Molecule CORM-3 Reveals Tricarboxylic Acid Cycle as Major TargetPublication . Carvalho, Sandra M.; Marques, Joana; Romão, Carlos C.; Saraiva, Lígia M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); American Society for MicrobiologyIn the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli. By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.
- Identification of the sirohaem biosynthesis pathway in Staphylococcus aureusPublication . Videira, Marco A.M.; Lobo, Susana A.L.; Sousa, Filipa L.; Saraiva, Lígia M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Federation of European Biochemical Societies | WileySirohaem is a modified tetrapyrrole and a key prosthetic group of several enzymes involved in nitrogen and sulfur metabolisms. This work shows that Staphylococcus aureus produces sirohaem through a pathway formed by three independent enzymes. Of the two putative sirohaem synthases encoded in the S. aureus genome and annotated as cysG, one is herein shown to be a uroporphyrinogen III methyltransferase that converts uroporphyrinogen III to precorrin-2, and was renamed as UroM. The second cysG gene encodes a precorrin-2 dehydrogenase that converts precorrin-2 to sirohydrochlorin, and was designated as P2D. The last step was found to be performed by the gene nirR that, in fact, codes for a protein with sirohydrochlorin ferrochelatase activity, labelled as ShfC. Additionally, site-directed mutagenesis studies of S. aureus ShfC revealed that residues H22 and H87, which are predicted by homology modelling to be located at the active site, control the ferrochelatase activity. Within bacteria, sirohaem synthesis may occur via one, two or three enzymes, and we propose to name the correspondent pathways as Types 1, 2 and 3, respectively. A phylogenetic analysis revealed that Type 1 is the most used pathway in Gammaproteobacteria and Streptomycetales, Type 2 predominates in Fibrobacteres and Vibrionales, and Type 3 predominates in Firmicutes of the Bacillales order. Altogether, we concluded that the current distribution of sirohaem pathways within bacteria, which changes at the genus or species level and within taxa, seems to be the result of evolutionary multiple fusion/fission events.
- The di-iron RIC protein (YtfE) of Escherichia coli interacts with the DNA-binding protein from starved cells (Dps) to diminish RIC protein-mediated redox stressPublication . Silva, Liliana S.O.; Baptista, Joana M.; Batley, Charlotte; Andrews, Simon C.; Saraiva, Lígia M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Molecular, Structural and Cellular Microbiology (MOSTMICRO); American Society for MicrobiologyThe RIC (repair of iron clusters) protein of Escherichia coli is a di-iron hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulfur clusters. In this work, we performed a bacterial two-hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified, and its potential interaction with RIC was tested by bacterial adenylate cyclase-based two-hybrid (BACTH) system, bimolecular fluorescence complementation, and pulldown assays. Using the activity of two Fe-S-containing enzymes as indicators of cellular Fe-S cluster damage, we observed that strains with single deletions of ric or dps have significantly lower aconitase and fumarase activities. In contrast, the ric dps double mutant strain displayed no loss of aconitase and fumarase activity with respect to that of the wild type. Additionally, while complementation of the ric dps double mutant with ric led to a severe loss of aconitase activity, this effect was no longer observed when a gene encoding a di-iron site variant of the RIC protein was employed. The dps mutant exhibited a large increase in reactive oxygen species (ROS) levels, but this increase was eliminated when ric was also inactivated. Absence of other iron storage proteins, or of peroxidase and catalases, had no impact on RIC-mediated redox stress induction. Hence, we show that RIC interacts with Dps in a manner that serves to protect E. coli from RIC protein-induced ROS.
- The di-iron RIC (YtfE) protein 1 of Escherichia coli interacts with the DNA-binding protein from starved cells (Dps) to diminish RIC-protein-mediated redox stressPublication . Silva, Liliana; Baptista, Joana; Bately, Charlotte; Andrews, Simon; Saraiva, LigiaThe RIC (Repair of Iron Clusters) protein of Escherichia coli is a di-iron hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulphur clusters. In this work, we performed a Bacterial Two Hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified and its potential interaction with RIC was tested by BACTH, Bimolecular-Fluorescence-Complementation and pull-down assays. (...)