Staphylococcus aureus: the paths and crosstalks that lead to heme

Tetrapyrroles are biological molecules widespread through all living organisms and necessary for fundamental cellular processes. Within the wide range of known tetrapyrroles, heme and siroheme are prosthetic groups in proteins responsible for important functions such as gas transport and storage, electron transport, cellular signaling, cellular detoxification as well as for reduction of sulfite and nitrite. Both molecules are synthesized from a last common tetrapyrrole precursor, namely uroporphyrinogen III, forming heme via four enzymatic steps know as the classical heme biosynthesis pathway and generating siroheme via an independent route. In some organisms such as sulfate and nitrate reducing bacteria, heme is alternatively synthesized through the siroheme-dependent pathway. (...

Identification of the sirohaem biosynthesis pathway in Staphylococcus aureus

Sirohaem 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.preprintpublishe

In Campylobacter jejuni, a new type of chaperone receives heme from ferrochelatase

Funding Information: JZ is a recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. FS acknowledges support from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation program (grant agreement 803768). This work was also financially supported by Fundação para a Ciência e Tecnologia (Portugal) through PTDC/BIA-BQM/28642/2017 grant (LS), the MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020 and UIDP/04612/2020), and the LS4FUTURE Associated Laboratory (LA/P/0087/2020). Publisher Copyright: Copyright © 2023 Zamarreño Beas, Videira, Karavaeva, Lourenço, Almeida, Sousa and Saraiva.Intracellular heme formation and trafficking are fundamental processes in living organisms. Bacteria and archaea utilize three biogenesis pathways to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (uro’gen III). In this study, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro’gen III into heme in Campylobacter jejuni, demonstrating that this bacterium utilizes the protoporphyrin-dependent (PPD) pathway. In general, limited knowledge exists regarding the mechanisms by which heme b reaches its target proteins after this final step. Specifically, the chaperones necessary for trafficking heme to prevent the cytotoxic effects associated with free heme remain largely unidentified. In C. jejuni, we identified a protein named CgdH2 that binds heme with a dissociation constant of 4.9 ± 1.0 µM, and this binding is impaired upon mutation of residues histidine 45 and 133. We demonstrate that C. jejuni CgdH2 establishes protein–protein interactions with ferrochelatase, suggesting its role in facilitating heme transfer from ferrochelatase to CgdH2. Furthermore, phylogenetic analysis reveals that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is the first protein identified as an acceptor of intracellularly formed heme, expanding our knowledge of the mechanisms underlying heme trafficking within bacterial cells.publishersversionpublishe

Desulfovibrio vulgarisCbiKPcobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC, that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP, which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter

Regulation of bacterial haem biosynthesis

Funding Information: This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) by grant PTDC/BIA-BQM/28642/2017 and through R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. We also acknowledge funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 810856. JB is recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. Funding Information: This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) by grant PTDC/BIA-BQM/28642/2017 and through R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. We also acknowledge funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 810856. JB is recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. Publisher Copyright: © 2021 Elsevier B.V.Haem 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.publishersversionpublishe