| 1. |
- Longo, Liam M., et al.
(författare)
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Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 117:10, s. 5310-5318
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in alpha beta alpha sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an alpha-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first alpha beta alpha sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.
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| 2. |
- Szeler, Klaudia
(författare)
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Computational Protein Evolution : Modeling the Selectivity and Promiscuity of Engineered Enzymes
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are biological catalysts that significantly increase the rate of all biochemical reactions that take place within cells and are essential to maintain life. Many questions regarding their function remain unknown. Experimental techniques, such as kinetic measurements, spectroscopy, and site-directed mutagenesis, can provide relevant information about enzyme structure, key residues, active site conformations, and kinetics. However, they struggle to provide a full picture of enzyme catalysis. Combining experiments with computational techniques gives the possibility to generate a complete explanation with atomistic resolution. Computational modeling offers an incredibly robust toolkit that can provide detailed insight into the reactivity and dynamics of biomolecules.Compounds that contain phosphate and sulfate groups are essential in the living world. They are present as i.e., a biological source of energy (ATP), signaling molecules (GTP), coenzymes, building blocks (DNA, RNA). Furthermore, phosphate esters can be used as insecticides, herbicides, flame retardants, and as chemical weapons. Cleavage of the phosphate bond involves an extremely low rate of spontaneous hydrolysis, nevertheless it is common reaction in living organisms. Phosphatases (enzymes catalysing cleavage of phosphate bond) are crucial in both physiological regulation as well as serious pathological conditions including asthma, immunosuppression, cardiovascular diseases, diabetes.Understanding the basis of phosphoryl and sulfuryl transfer reactions is crucial for medical, biological, and biotechnological industries in order to i.e., create and improve existing drugs, modify enzyme structures, understand the development of some diseases. However, despite decades of both experimental and computational studies, mechanistic details of these reactions remain controversial. These reactions can occur via multiple different mechanisms involving intermediate steps or transition state structures. To solve these puzzles, we performed computational studies to verify the reaction pathway of diaryl sulfate diesters hydrolysis. We suggest that the reaction proceeds through a concerted mechanism with a loose (slightly dissociative) transition state.Serum paraoxonase 1 (PON1) is calcium-dependent lactonase, which is bound to high-density lipoprotein (HDL) with apolipoprotein A-I (ApoA-I). The enzyme is highly promiscuous and catalyzes the hydrolysis of multiple, different types of chemical compounds, such as lactones, aromatic esters, oxons, and organophosphates. We performed several, complex studies on PON1’s reaction mechanism, promiscuity, PON1-HDL interactions, and evolutionary trajectories. One of the most extensively used approaches in this thesis was the empirical valence bond (EVB) method. Our models reproduce essential experimental observables and provide mechanistic insights and a better understanding of the enzymes role and its evolutionary derived promiscuity.
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| 3. |
- Baier, Florian, et al.
(författare)
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Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes
- 2019
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-beta-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.
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| 4. |
- Barrozo, Alexandre, et al.
(författare)
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Computer simulations of the catalytic mechanism of wild-type and mutant beta-phosphoglucomutase
- 2018
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 16:12, s. 2060-2073
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Tidskriftsartikel (refereegranskat)abstract
- beta-Phosphoglucomutase (beta-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of beta-glucose-1-phosphate to -glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of beta-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.
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| 5. |
- Ben-David, Moshe, et al.
(författare)
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Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition
- 2020
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:4, s. 1133-1147
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Tidskriftsartikel (refereegranskat)abstract
- Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
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| 6. |
- Biler, Michal, et al.
(författare)
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Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase
- 2020
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Ingår i: Journal of the American Chemical Society. - : AMER CHEMICAL SOC. - 0002-7863 .- 1520-5126. ; 142:47, s. 20216-20231
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics simulations were performed on wild-type AMDase and variants thereof to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the nonconverted or poorly converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.
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| 7. |
- Braida, Benoit, et al.
(författare)
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Preface
- 2017
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Ingår i: Computational and Theoretical Chemistry. - : Elsevier BV. - 2210-271X .- 2210-2728. ; 1116:SI, s. 1-1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 8. |
- Brickel, Sebastian, et al.
(författare)
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Q-RepEx : A Python pipeline to increase the sampling of empirical valence bond simulations
- 2023
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Ingår i: Journal of Molecular Graphics and Modelling. - : Elsevier. - 1093-3263 .- 1873-4243. ; 119
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Tidskriftsartikel (refereegranskat)abstract
- The exploration of chemical systems occurs on complex energy landscapes. Comprehensively sampling rugged energy landscapes with many local minima is a common problem for molecular dynamics simulations. These multiple local minima trap the dynamic system, preventing efficient sampling. This is a particular challenge for large biochemical systems with many degrees of freedom. Replica exchange molecular dynamics (REMD) is an approach that accelerates the exploration of the conformational space of a system, and thus can be used to enhance the sampling of complex biomolecular processes. In parallel, the empirical valence bond (EVB) approach is a powerful approach for modeling chemical reactivity in biomolecular systems. Here, we present an open-source Python-based tool that interfaces with the Q simulation package, and increases the sampling efficiency of the EVB free energy perturbation/umbrella sampling approach by means of REMD. This approach, Q-RepEx, both decreases the computational cost of the associated REMD-EVB simulations, and opens the door to more efficient studies of biochemical reactivity in systems with significant conformational fluctuations along the chemical reaction coordinate.
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| 9. |
- Burke, Jason R., et al.
(författare)
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Bifunctional Substrate Activation via an Arginine Residue Drives Catalysis in Chalcone Isomerases
- 2019
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 9:9, s. 8388-8396
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Tidskriftsartikel (refereegranskat)abstract
- Chalcone isomerases are plant enzymes that perform enantioselective oxa-Michael cyclizations of 2'-hydroxychalcones into flavanones. An X-ray crystal structure of an enzyme-product complex combined with molecular dynamics simulations reveal an enzyme mechanism wherein the guanidinium ion of a conserved arginine positions the nucleophilic phenoxide and activates the electrophilic enone for cyclization through Bronsted and Lewis acid interactions. The reaction terminates by asymmetric protonation of the carbanion intermediate syn to the guanidinium. Interestingly, bifunctional guanidine- and urea-based chemical reagents, increasingly used for asymmetric organocatalytic applications, share mechanistic similarities with this natural system. Comparative protein crystal structures and molecular dynamics simulations further demonstrate how two active site water molecules coordinate a hydrogen bond network that enables expanded substrate reactivity for 6'-deoxychalcones in more recently evolved type-2 chalcone isomerases.
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| 10. |
- Calixto, Ana R., et al.
(författare)
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GTP Hydrolysis Without an Active Site Base : A Unifying Mechanism for Ras and Related GTPases
- 2019
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Ingår i: Journal of the American Chemical Society. - : AMER CHEMICAL SOC. - 0002-7863 .- 1520-5126. ; 141:27, s. 10684-10701
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Tidskriftsartikel (refereegranskat)abstract
- GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the G(alpha i), subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.
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