Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase
Guilherme Vilela-Alves, Rita Rebelo Manuel, Neide Pedrosa, Ines A. Cardoso Pereira, Maria Joao Romao and Cristiano Mota [1]
Molecular Tour
Mo/W-dependent Formate dehydrogenases (Fdhs) are enzymes that catalyze the reversible interconversion of CO2 into formate. They are promising targets for optimization, due to their efficiency, selectivity and high activity; as well as inspiration for the engineering of industrial bio-inorganic catalysts to reduce CO2 into added-value compounds.
Mo/W-Fdhs present varied quaternary structures around the conserved catalytic α subunit, allowing for interactions with a large assortment of electron acceptors/mediators located in membranes or soluble in the cytoplasm and periplasm.
The catalytic subunit from Desulfovibrio vulgaris FdhAB contains a W center, coordinating two Molybdopterin Guanine Dinucleotides (MGD), one terminal sulfido ligand (-SH/=S) and a SeCys (selenocysteine) residue from the polypeptide chain, in a distorted trigonal prismatic geometry. In the second coordination sphere of the metal, two conserved residues are catalytically relevant: a histidine, thought to play a role in proton transfer, and an arginine involved in substrate orientation and stabilization of putative catalytic intermediates [2].
A recently discovered disulphide redox switch was shown to be crucial in an allosteric mechanism for enzyme activation (yielding maximum activity) or inactivation (resulting in protection against O2 damage) [3]. This allosteric mechanism makes use of conformational changes that extend from the surface exposed disulfide bond towards the deeply buried residue M405 near the active site, adopting new conformations.
In a previous work[3], the M405A mutation virtually abolished the catalytic activity, and its structure revealed a significantly distortion of the active site, particularly the protein backbone near SeCys192 (U192), preventing the modeling of U192 side chain and the understanding of its possible catalytic role. Comparison between and active sites. In this work, the M405S mutation was used to probe the prominent role of M405 on the metal site geometry, as we were able to fully model the W site geometry of this variant. stabilized the M405S mutant when compared to M405A. The side chain of Ser405 is shown, the Ser405 Oγ–W distance and the hydrogen bonds established to O2α and O2β from the phosphate groups of MGD1 are shown as dashed lines. Thus, we could confirm, in M405S, the significant rearrangement and increased mobility of the I191-T196 helical region (that contains the mechanistically relevant residues U192 and H193), caused by the absence of the bulky M405 side chain, as well as its impact on the flexibility and geometry of the active site. Effect of the absence of the Met405 side chain on the B factors of the Cα atoms of Ile191–Thr196 of DvFdhAB:
- .
- (PDB entry 8cm7). The loop Gly896–Ile992, was not modelled for this data set.
- (PDB entry 6sdr).
In all scenes, the tungsten active site and the proximal [4Fe–4S] cluster are shown as sticks and spheres, the peptide chain is shown in ribbon representation. B factors were normalized for each structure independently. .
References
- ↑ Vilela-Alves G, Rebelo Manuel R, Pedrosa N, Cardoso Pereira IA, Romão MJ, Mota C. Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase. Acta Crystallogr F Struct Biol Commun. 2024 May 1. PMID:38699971 doi:10.1107/S2053230X24003911
- ↑ doi: https://dx.doi.org/10.1021/acscatal.0c00086
- ↑ 3.0 3.1 Oliveira AR, Mota C, Vilela-Alves G, Manuel RR, Pedrosa N, Fourmond V, Klymanska K, Léger C, Guigliarelli B, Romão MJ, Cardoso Pereira IA. An allosteric redox switch involved in oxygen protection in a CO(2) reductase. Nat Chem Biol. 2024 Jan;20(1):111-119. PMID:37985883 doi:10.1038/s41589-023-01484-2
