Mota, C, Webster M, Saidi M, Kapp U, Zubieta C, Giachin G, Manso JA, de Sanctis D.
2024.
Metal ion activation and DNA recognition by the Deinococcus radiodurans manganese sensor DR2539. bioRxiv. : Cold Spring Harbor Laboratory
AbstractThe accumulation of manganese ions is crucial for scavenging reactive oxygen species (ROS) and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains: a DNA binding domain, a dimerization domain, and an ancillary domain, both the metal ion activation mechanism and the DNA recognition mechanism remain elusive. In this study, we present biophysical analyses and the structure of the dimerization and DNA binding domains of DR2539 in its holo form and in complex with the 21 bp pseudo-palindromic repeat of the dr1709 promotor region. These findings shed light into the activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix H3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small angle X-ray scattering experiments and supported by AlphaFold modelling provides a model illustrating the conformational changes induced upon metal binding.Competing Interest StatementThe authors have declared no competing interest.
Vilela-Alves, G, Manuel RR, Viegas A, Carpentier P, Biaso F, Guigliarelli B, Pereira IAC, Romão MJ, Mota C.
2024.
Substrate-dependent oxidative inactivation of a W-dependent formate dehydrogenase involving selenocysteine displacement. bioRxiv. : Cold Spring Harbor Laboratory
AbstractMetal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards novel strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be fully understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the O2 sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a new species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This new form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyze the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate. These results reveal that oxidative inactivation does not require reduction of the metal, as widely assumed, as it can also occur in the oxidized state in the presence of CO2.Competing Interest StatementThe authors have declared no competing interest.AORAldehyde Oxido-reductaseDTTDithiothreitolDvDesulfovibrio vulgarisEPRElectron Paramagnetic ResonanceFdhFormate dehydrogenaseHPHigh PressureMGDMolybdopterin Guanine DinucleotidesNDNew dropROSReactive Oxygen SpeciesSODSuperoxide dismutaseTSAThermal Shift Assay