Oliveira, AR, Mota C, Mourato C, Domingos RM, Santos MFA, Gesto D, Guigliarelli B, Santos-Silva T, Romão MJ, Pereira IAC.
2020.
Towards the mechanistic understanding of enzymatic CO2 reduction, 2020. ACS CatalysisACS Catalysis. : American Chemical Society
AbstractReducing CO2 is a challenging chemical transformation that biology solves easily, with high efficiency and specificity. In particular, formate dehydrogenases are of great interest since they reduce CO2 to formate, a valuable chemical fuel and hydrogen storage compound. The metal-dependent formate dehydrogenases of prokaryotes can show high activity for CO2 reduction. Here, we report an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough fully loaded with cofactors, its cata-lytic characterization and crystal structures in oxidised and reduced states. The enzyme has very high activi-ty for CO2 reduction and displays remarkable oxygen stability. The crystal structure of the formate-reduced enzyme shows Sec still coordinating the tungsten, supporting a mechanism of stable metal coordination during catalysis. Comparison of the oxidised and reduced structures shows significant changes close to the active site. The DvFdhAB is an excellent model for studying catalytic CO2 reduction and probing the mecha-nism of this conversion.Reducing CO2 is a challenging chemical transformation that biology solves easily, with high efficiency and specificity. In particular, formate dehydrogenases are of great interest since they reduce CO2 to formate, a valuable chemical fuel and hydrogen storage compound. The metal-dependent formate dehydrogenases of prokaryotes can show high activity for CO2 reduction. Here, we report an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough fully loaded with cofactors, its cata-lytic characterization and crystal structures in oxidised and reduced states. The enzyme has very high activi-ty for CO2 reduction and displays remarkable oxygen stability. The crystal structure of the formate-reduced enzyme shows Sec still coordinating the tungsten, supporting a mechanism of stable metal coordination during catalysis. Comparison of the oxidised and reduced structures shows significant changes close to the active site. The DvFdhAB is an excellent model for studying catalytic CO2 reduction and probing the mecha-nism of this conversion.
Vilela-Alves, G, Manuel RR, Oliveira AR, Pereira IC, Romão MJ, Mota C.
2023.
Tracking W-Formate Dehydrogenase Structural Changes During Catalysis and Enzyme Reoxidation. International Journal of Molecular Sciences. 24, Number 1
AbstractMetal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.
Otrelo-Cardoso, AR, Nair RR, Correia MAS, Rivas MG, Santos-Silva T.
2014.
TupA: A Tungstate Binding Protein in the Periplasm of Desulfovibrio alaskensis G20, 2014/05/29/accep. International Journal of Molecular Sciences. 15(7):11783-11798.: MDPI
AbstractThe TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P2(1) space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.