Dataset Information

Identification and characterization of Formate dehydrogenases (FDHs) from Bacillus subtilis

ABSTRACT: The Mo/W-bisPGD Formate Dehydrogenases (FDHs) family interplays in several metabolic pathways ranging from carbon fixation to energy harvesting owing to their reaction with a wide variety of redox partners. Here, we have unveiled two non-canonical FDHs in Bacillus subtilis which are organized into two-subunit complexes, as shown by mass spectrometry, with unique features, ForCE1 and ForCE2. The ForC catalytic subunit interacts with an unprecedented partner subunit ForE and its amino acid sequence within the active site deviates from the consensus residues typically associated to FDH activity, with an histidine residue naturally substituted by a glutamine. The ForE essential subunit mediates the utilization of menaquinone as electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency EPR experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature consistent with the characteristic Mo-bisPGD cofactor of this enzyme family. Altogether, this work enlarges our representation of the FDH family by deciphering a non-canonical FDH, which differs both in terms of architecture, amino acid conservation around the Mo cofactor and reactivity.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bacillus Subtilis Subsp. Subtilis Str. 168

SUBMITTER: Regine Lebrun

LAB HEAD: Regine Lebrun

PROVIDER: PXD028742 | Pride | 2022-02-16

REPOSITORIES: Pride

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Publications

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

Arias-Cartín Rodrigo R Uzel Alexandre A Seduk Farida F Gerbaud Guillaume G Pierrel Fabien F Broc Marianne M Lebrun Régine R Guigliarelli Bruno B Magalon Axel A Grimaldi Stéphane S Walburger Anne A

The Journal of biological chemistry 20211106 2

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are orga ...[more]

PMID: 34748728

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Project description:The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions and its induction is controlled by two histidine kinases, DosS and DosT. Recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. This report demonstrates that DosS responds to a reduced electron transport system (ETS), although this does not rule out a role for oxygen in silencing signaling. Under aerobic conditions induction of the DosR regulon by DosS but not DosT was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating the menaquinone pool is a limiting factor in regulon induction. Taken together these data indicate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS, rescue inhibition of menaquinone synthesis, and is less toxic. The menaquinone pool is central to the ETS and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Aerobically growing logarithmic phase DosS and DosT mutant strains were analyzed after treatment with the cytochrome c reductant ascorbate to examine the effect on DosR signaling caused by reducing the electron transport system. Experiments were repeated in triplicate (dosT mutant) or duplicate (dosS mutant).

Dataset Information

Identification and characterization of Formate dehydrogenases (FDHs) from Bacillus subtilis

Publications

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

Similar Datasets

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