Browsing by Author "Gomez Llanos, Aharon Ignacio"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Difusión y unión de CO2 en la enzima Crotonil-CoA Carboxilasa-Reductasa mediante simulaciones de dinámica molecular.
    (Universidad de Concepción., 2023) Gomez Llanos, Aharon Ignacio; Vöhringer Martínez, Esteban
    Carbon dioxide (CO2) is one of the most important greenhouse gases, and its accumulation in the atmosphere is associated with climate change. The fixation of this gas using enzymes for the generation of value-added organic compounds presents a current challenge in biocatalysis. Crotonyl-CoA Carboxylase-Reductase (Ccr) is one of the most efficient enzymes in CO2 fixation, showing the highest catalytic rate constant and being insensitive to the presence of oxygen, unlike RuBisCO, which is the enzyme responsible for CO2 fixation in nature. This thesis studied the conformational changes of Ccr to understand its high efficiency. Ccr is a homo-tetramer that, in the presence of NADPH and substrate analogs (substrate, carboxylation or reduction product), undergoes conformational changes, transitioning from a completely symmetric structure to a dimer of dimers configuration. Each of these dimers has an open subunit without substrate and a closed one with substrate where the reaction occurs. Its high catalytic efficiency is believed to be associated with these conformational changes leading to half site reactivity. Molecular dynamics simulations were performed to study the conformational changes of Ccr and CO2 binding. A method was developed to analyze the effect of subunit conformation on CO2 concentration at the active site, thus defining CO2 binding sites. Additionally, a procedure was developed to quantify the average residence time of CO2 molecules in the previously defined binding sites.With the combined information on conformational changes, binding, and residence of CO2 at the active site, a catalytic mechanism for the enzyme was proposed. Initially, molecular dynamics simulations were conducted to determine the equilibrium conformations of the system and possible changes in these conformations in aqueous solution. These simulations showed that the presence of the substrate, in its reactive position, maintains the closed conformation of the active site. In the absence of the substrate, the subunit undergoes an opening conformational change on the scale of tens of nanoseconds. Three residues in enoyl carboxylase-reductase (ECR) enzymes from primary metabolism (E151, N218 y N157) are conserved in Ccr. Compared to ECRs from the secondary metabolism, where these residues are not conserved, the catalytic activity is reduced, on average, 20 times. These residues are at the interface between both dimers and are far from the active site. Disrupting the interaction of these residues through point mutations in Ccr decreases its catalytic efficiency by three orders of magnitude. Principal component analysis of the mutant simulations indicates that the opening of the active site becomes slower, suggesting a direct relationship between conformational changes and catalytic efficiency. To study the effect of conformation on CO2 concentration at the active site, simulations were performed with explicit CO2 molecules in representative Ccr conformations. The distributions of CO2 molecules and their relative concentrations at the active site were analyzed for wild-type Ccr and its nonreactive variant N81L. A computational method was developed to define the binding sites, which can serve as an initial approximation to a Markov state model. The estimation of relative concentration and the definition of binding sites were validated comparing with experimental information. The results demonstrate a conformational dependence of CO2 concentration at the active site. In the absence of substrate, the highest affinity is found in the closed active site. The presence of substrate in the closed active site reverses the affinity of the subunits of the dimer, with the open conformation presenting a higher affinity at its active site. Substrate presence in the open subunit reduces its affinity by one-third without altering the closed site’s affinity with substrate. When comparing the wild-type system with the N81L variant, the subunits’ affinity remained unmodified. However, binding sites were not detected in the open active site close to the residues, the substrate and cofactor associated with the chemical reaction. This findings explained the experimentally observed loss of activity for the carboxylation of this mutant. The previously defined binding sites were used to study the kinetics of CO2 binding in the elementary steps of catalysis and model the diffusion of CO2 molecules to the protein’s active site. The closed conformation of the subunit with substrate does not present an access pathway for CO2 to reach the active site. In the open subunit, there are binding sites associated with residues important for the carboxylation reaction. These sites are conserved for the open conformation of the subunit, regardless of the substrate’s presence, and have average residence times on the nanosecond scale. The residence times in these binding sites are modified by the presence of substrate, favoring those that facilitate carboxylation, allowing CO2 to remain bound during the conformational change for closing active site and subsequent reaction. In summary, the results obtained in this thesis allowed proposing a mechanism for the enzyme’s catalysis. In the first step, CO2 binds to the open subunit without substrate. Subsequently, substrate binding occurs, positioning CO2 for the reaction and triggering the conformational change. Finally, the chemical reaction takes place. This work provides an atomic-molecular view of Ccr’s function in CO2 fixation catalysis, enabling the proposal of mutations capable of improving CO2 availability at the active site. Additionally, based on the results obtained, a catalytic cycle was proposed to explain the enzyme’s high efficiency. Each subunit in the tetrameric structure fulfills a distinct function maximizing the efficiency of the different stages of the cycle.
Síguenos en...