Biology subjects

Osuna, S.

Publications and source records attributed to Osuna, S..

10 recordsLinked to original sources

bioRxiv2026

Substituting a conserved histidine alleviates difluoroacetate-dependent isoaspartate formation in a fluoroacetate dehalogenase

Organofluorides are ubiquitous in modern society, but their persistence poses significant environmental and health risks. Fluoroacetate dehalogenases (FAcDs) are promising candidates for the bioremediation of polyfluorinated substances, as they can hydrolyze C-F bonds efficiently under mild conditions. However, their activity toward gem-difluorinated carboxylic acids, such as 2,2-difluoroacetate (F2A), is typically poor, with rates orders of magnitude lower than for monofluorinated substrates (e.g. fluoroacetate). Here, we combine kinetic studies, mass spectrometric and biochemical analyses, and computational modeling to pinpoint the F2A-induced isomerization of the catalytic aspartate (Asp105) as a major inactivation pathway that severely limits sustained F2A turnover. To overcome this inhibition mechanism, we discovered that a highly conserved histidine (His104) acts as a gatekeeper for sustained F2A activity. Substituting His104 with the noncanonical N3-methylhistidine increased activity 14-fold, while replacement with asparagine boosted activity almost 50-fold, yielding a potent F2A defluorinase with an initial degradation rate of 0.3 s-1. Computational studies elucidated that these substitutions reshape the conformational landscape of Asp105, suppressing isoAsp-promoting conformations while preserving catalytically productive poses for F2A conversion. Notably, transplanting the His-to-Asn substitution into two homologous FAcDs drastically reduced their activity, highlighting H1 as a uniquely suitable scaffold for engineering promiscuous F2A conversion. Overall, this study demonstrates that targeting mechanistic intricacies can significantly enhance FAcD performance on F2A, providing a foundation for further engineering of H1 to degrade polyfluorinated pollutants.

biochemistry↗

bioRxiv2025

Boosting reversible photocontrol of a photoxenase by an engineered conformational shift

Our study successfully explores strategies to effectively improve the photocontrol efficiency of light-sensitive enzymes, dubbed photoxenases, with photoswitchable unnatural amino acids (UAAs). The engineering of photoxenases is a versatile method for the reversible photocontrol in various applications. To boost the photocontrol of an established allosteric and heterodimeric photoxenase based on imidazole glycerol phosphate synthase, we turned from an ineffective tuning of the UAA photochemistry to a semi-rational enzyme design. Remarkably, mutations at the catalytically important heterodimer interface increased the light-regulation factor (LRF) for the kcat up to ~100 with near-quantitative reversibility. Steady-state kinetic investigations combined with computationally determined correlation-based Shortest-Path-Map analysis and conformational landscapes revealed how photocontrol was altered in the two best hits. The LRF(kcat) correlated with a shift of a conformational equilibrium between an active and inactive population at the targeted active site and a tuned population productivity upon irradiation. While the overall reduced kcat values originated from a rewiring of the allosteric signal transmission, the increased LRF(kcat) resulted from a change in i) the size of the conformational shift, ii) the population productivity, and iii) the conformational heterogeneity. With this, our findings provide initial guidelines to boost photocontrol and underscore the power of photoxenase engineering.

biochemistry↗

bioRxiv2025

Expanding the Repertoire of Photoswitchable Unnatural Amino Acids for Enzyme Engineering

Photoswitchable unnatural amino acids (psUAAs) play a crucial role in the engineering of light-sensitivity in enzymes, which holds significant promise for diverse applications such as biotherapy and biocatalysis. Besides near-quantitative photoconversion, the success and expediency of a psUAA for a certain application is defined by its interaction potential with the enzyme, its thermal stability and its effective wavelength of irradiation. To establish high versatility in the current repertoire, we have designed and synthesized six psUAAs based on azobenzene, arylazopyrazole, arylazothiazole, hemithioindigo and spiropyran photoswitches. The resulting psUAAs exhibit an enhanced interaction potential within an enzyme owing to their capacity for hydrogen bonding, ionic interactions, and metal ion coordination. Moreover, we observed diverse photochemical behaviors among the psUAAs, with four of them reversibly switching between the isomers with purely visible light. Notably, we identified orthogonal aminoacyl-tRNA synthetases that facilitate the incorporation of five of the six psUAAs co-translationally and computationally analyzed the synthetase-psUAA interactions. Finally, we evaluated the photochemical behavior of the five psUAAs within an enzymatic model and tested the photocontrol of catalysis confirming their diversity. Ultimately, our findings significantly expanded the repertoire of psUAAs and demonstrated their feasibility for enzyme engineering studies.

synthetic biology↗

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 enhance catalysis in designed enzymes by facilitating substrate binding and product release

The role of amino-acid residues distant from an enzymes active site in facilitating the complete catalytic cycle--including substrate binding, chemical transformation, and product release-- remains poorly understood. Here, we investigate how distal mutations promote the catalytic cycle by engineering mutants of three de novo Kemp eliminases containing either active-site or distal mutations identified through directed evolution. Kinetic analyses, X-ray crystallography, and molecular dynamics simulations reveal that while active-site mutations create preorganized catalytic sites for efficient chemical transformation, distal mutations enhance catalysis by facilitating substrate binding and product release through tuning structural dynamics to widen the active-site entrance and reorganize surface loops. These distinct contributions work synergistically to improve overall activity, demonstrating that a well-organized active site, though necessary, is not sufficient for optimal catalysis. Our findings reveal critical roles that distal residues play in shaping the catalytic cycle to enhance efficiency, yielding valuable insights for enzyme design.

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↗

bioRxiv2023

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

bioengineering↗

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↗

bioRxiv2020

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. Yet such efforts are rare and focus only on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report the first multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We developed a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative sign and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and beta-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers lessons for engineering P450s.

evolutionary biology↗

bioRxiv2020

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP+

Efficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usual specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity towards NADP+, and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

biochemistry↗