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 catalyse 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 centre, 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 [1].
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) [2]. 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[2], 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 modelling of U192 side chain and the understanding of its possible catalytic role. 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. The hydrogen bonding between the mutated serine (S405 in the mutant) and the two phosphate groups from MGD1 stabilized the M405S mutant when compared to M405A. 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.
References:
[1] A. R. Oliveira et al., “Toward the Mechanistic Understanding of Enzymatic CO2 Reduction,” ACS Catal, vol. 10, no. 6, pp. 3844–3856, 2020, doi: 10.1021/acscatal.0c00086.
[2] A. R. Oliveira et al., “An allosteric redox switch involved in oxygen protection in a CO2 reductase,” Nat Chem Biol, no. 20, pp. 111–119, Nov. 2024, doi: 10.1038/s41589-023-01484-2.
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
