Biology subjects

Feixas, F.

Publications and source records attributed to Feixas, F..

5 recordsLinked to original sources

bioRxiv2025

Single-Molecule Kinetic Exploration of Functional Substates in an Evolving Phosphotriesterase

Enzymes achieve catalysis by dynamically sampling diverse conformational states. Beyond this plasticity, individual enzyme molecules occupy metastable substates, forming an ensemble of functional substates within a population. Since shifts in functional substate dynamics drive phenotypic variation, their evolutionary trajectories are central to the emergence of new functions. However, the challenge of measuring functional substates has hindered our understanding of their role in enzyme evolution and the optimization of conformational substates. Here, we address this gap by investigating how functional and conformational substates were modulated during enzyme functional transitions, using single-molecule kinetic assays and molecular dynamics simulations. We analyzed wild-type phosphotriesterase (PTE) and 18 evolved variants that transitioned from the native PTE to promiscuous arylesterase (AE) activity. Our findings reveal that evolutionary transitions reshape functional and conformational substate landscapes: PTE-specialized variants exhibit broader substate distributions, whereas AE-specialized variants display more uniform substates. These results provide the first direct evidence that enzyme evolution is accompanied by coordinated shifts in functional and conformational substate equilibria, optimizing both for the enzymes catalytic efficiency. This work highlights the power of single-molecule techniques in uncovering how heterogeneous enzyme populations navigate substate transitions and, ultimately, how these transitions shape enzyme evolvability.

biochemistry↗

bioRxiv2025

Distal mutations in a designed retro-aldolase alter loop dynamics to shift and accelerate the rate-limiting step

Amino-acid residues distant from an enzymes active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.

biochemistry↗

bioRxiv2023

Origin Of Evolutionary Bifurcation In An Enzyme

Evolution can lead to significantly distinct outcomes depending on the mutational path taken. Evolutionary bifurcation, in which two mutational trajectories segregate, becoming non-interchangeable over time, is the basis of diversification in all kingdoms of life. Here, we present a detailed molecular description of a bifurcation event that rapidly led to the emergence of two distinct enzymes from a common ancestor. When initiated from two starting points that differed by a single amino acid, the laboratory evolution of a phosphotriesterase (PTE) toward arylester hydrolysis resulted in different genetic and phenotypic outcomes. One trajectory led to a >35,000-fold increase in activity via the reorganization of its active site to achieve exquisite enzyme-substrate complementarity. The second trajectory gave rise to an evolved variant with a [~]500-fold increase in activity, but exhibiting an alternative substrate binding mode resulting from the destabilization of an active site loop. While initial mutations tend to dictate mutational accessibility, we rather observed the gradual divergence and specialisation of each trajectory, following the emergence of distinct molecular interaction networks. Intramolecular epistasis underlay pathway bifurcation by promoting unique synergistic interactions within each trajectory, while restricting the fixation of mutation across pathways. Our results illustrate how distinct molecular outcomes can radiate from a common protein ancestor and give rise to phenotypic diversity.

evolutionary biology↗

bioRxiv2022

Changes in protonation states of in-pathway residues can alter ligand binding pathways obtained from spontaneous binding molecular dynamics simulations

Protein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 [A] away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations.

biophysics↗

bioRxiv2021

Unravelling the Graded Millisecond Allosteric Activation Mechanism of Imidazole Glycerol Phosphate Synthase

Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of key functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond timescale interconversion between these functional states hamper their detailed experimental and computational characterization. Here, we design a computational strategy tailored to reconstruct millisecond timescale events to describe the graded allosteric activation of imidazole glycerol phosphate synthase (IGPS) in the ternary complex. IGPS is a heterodimeric bienzyme complex responsible for the hydrolysis of glutamine to glutamate in the HisH subunit and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation pathway of wild-type IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that effector binding in HisF dramatically decreases the conformational barrier associated with the oxyanion hole formation in HisH, in line with the experimentally observed 4500-fold activity increase in glutamine production. The formation of the allosterically active state is controlled by time-evolving dynamic communication networks connecting the effector and substrate binding sites. This computational strategy can be generalized to study other unrelated enzymes undergoing millisecond timescale allosteric transitions.

biophysics↗