33 research outputs found
Carbon Dioxide Utilisation -The Formate Route
UIDB/50006/2020 CEEC-Individual 2017 Program Contract.The relentless rise of atmospheric CO2 is causing large and unpredictable impacts on the Earth climate, due to the CO2 significant greenhouse effect, besides being responsible for the ocean acidification, with consequent huge impacts in our daily lives and in all forms of life. To stop spiral of destruction, we must actively reduce the CO2 emissions and develop new and more efficient “CO2 sinks”. We should be focused on the opportunities provided by exploiting this novel and huge carbon feedstock to produce de novo fuels and added-value compounds. The conversion of CO2 into formate offers key advantages for carbon recycling, and formate dehydrogenase (FDH) enzymes are at the centre of intense research, due to the “green” advantages the bioconversion can offer, namely substrate and product selectivity and specificity, in reactions run at ambient temperature and pressure and neutral pH. In this chapter, we describe the remarkable recent progress towards efficient and selective FDH-catalysed CO2 reduction to formate. We focus on the enzymes, discussing their structure and mechanism of action. Selected promising studies and successful proof of concepts of FDH-dependent CO2 reduction to formate and beyond are discussed, to highlight the power of FDHs and the challenges this CO2 bioconversion still faces.publishersversionpublishe
Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase
The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzyme-catalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using two-dimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (). We observe that the frequency–frequency time correlation functions (FFCF) for the ternary complexes with NAD+ and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window
3-Picolyl Azide Adenine Dinucleotide as a Probe of Femtosecond to Picosecond Enzyme Dynamics
Hybrid Quantum and Classical Simulations of the Formate Dehydrogenase Catalyzed Hydride Transfer Reaction on an Accurate Semiempirical Potential Energy Surface
A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase
The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of E. coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between those two crystal structures is in the anisotropic B-factors, which indicates disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature-dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and acceptor at the tunneling ready state. These observations suggest that that the mutation disrupts the concerted motions that bring the H-donor and acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different timescales on the hydride transfer reaction within a complex enzymatic mechanism
Infrared spectroscopy of nicotinamide adenine dinucleotides in one and two dimensions
The development of multidimensional spectroscopic tools capable of resolving site-specific information about proteins and enzymes in the solution phase is an important aid to our understanding of biomolecular mechanisms, structure, and dynamics. Nicotinamide adenine dinucleotide (NAD) is a common biological substrate and so offers significant potential as an intrinsic vibrational probe of protein-ligand interactions but its complex molecular structure and incompletely characterized infrared spectrum currently limit its usefulness. Here, we report the FTIR spectroscopy of the oxidized and reduced forms of NAD at a range of pD values that relate to the "folded" and "unfolded" forms of the molecules that exist in solution. Comparisons with structural analogs and the use of density functional theory simulations provide a full assignment of the observed modes and their complex pD dependencies. Finally, ultrafast two-dimensional infrared spectra of the oxidized and reduced forms of NAD are reported and their usefulness as biomolecular probes is discussed
Mg 2+
Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e. the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg(2+) has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg(2+) binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg(2+) near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg(2+) binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg(2+) to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg(2+) accelerates the hydride transfer by ca. 7-fold but does not affect the magnitude or temperature-dependence of the intrinsic kinetic isotope effect. These results suggest that Mg(2+) facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg(2+) concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg(2+) with the glutamyl-tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with poly-glutamyl substitutes as species-specific antibiotic drugs