59 research outputs found
Computational library design and screening: Creating elephant paths in enzyme evolution
Traditional directed evolution employs iterations combining mutagenesis to generate genetic diversity and phenotypic selection to find improved variants. The final result typically consists of enzyme variants carrying multiple mutations, sometimes replacing up to 15% of the amino acids, and such variants can exhibit highly attractive properties. The improvements must be based on biophysical principles, such as improved hydrophobic packing and long range electrostatic interactions that account for better stability, removal of steric clashes to broaden substrate range, or modified electrostatics to change pH optima. Since many of these biophysical principles are known and can be modeled with computational algorithms, it is challenging to pursue the replacement of laborious and time-consuming directed evolution protocols by computational workflows. This is especially attractive if the desired properties cannot be screened in a high-throughput format, e.g. due to the complexity of an expression system or the lack of miniaturized assays. Accordingly, we are exploring the use of computational protein design, docking simulations and molecular dynamics simulations of mutant enzyme libraries to develop enzyme engineering strategies in which most of the laboratory screening is replaced by in-silico methods. We improved thermostability (ΔTm,app +22-35ºC) and cosolvent (DMSO, DMF, methanol) compatibility of an epoxide hydrolase, two dehalogenases and a peptide amidase using such a computational design and screening strategy (FRESCO, framework for rapid enzyme stabilization by computational library design). We also examined the use of a computational workflow (CASCO, catalytic selectivity by computation) that included active site redesign and molecular dynamics simulations on large numbers of mutant enzymes to predict multi-site mutants with controlled substrate selectivity. These were made in a single step to give highly enantioselective and enantiocomplementary epoxide hydrolases for use in the conversion of meso substrates to enantioenriched diols as well as enantioselective hydrolases for use in biocatalytic kinetic resolutions.
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Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design
Additional file 4: Figure S1. Michaelis–Menten graph of WT and 7xHMFO. Kinetic assay performed with HRP peroxidase in 50 mM phosphate buffer pH 8.0 at 25 °C using HMF as substrate
Computational redesign of transaminase active site
Aminotransferases are widely exploited in simple as well as more elaborate multi-enzymatic cascade reactions as an environmentally friendly alternative to transition metal catalysis. However, efficient selective conversion of numerous targets is a great limitation to date [1]. Attempts to improve substrate scope have been undertaken by generation and screening of large mutant libraries, which is very time-consuming and raises costs concerns [2]. Recent approaches explored the use of molecular docking of demanding substrates, followed by energy minimization and/or MD simulations [1;3]. Still, the best results have been obtained by extensive mutagenesis and screening.
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Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering
The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an α/β-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in (Formula presented.) of >2 °C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC-G40K-A134L-G220D-Y238W and AqdC-G40K-G220D-Y238W showed extended half-lives and the best trade-off between stability and activity, with increases in (Formula presented.) of 11.8 and 6.1 °C and relative activities of 22 and 72 %, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site
Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations
ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants
Identification and characterization of archaeal and bacterial F420-dependent thioredoxin reductases
The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as reducing cofactor. Yet, in 2016 a new type of thioredoxin reductase was discovered in archaea which utilizes instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that dependent on NADPH (NTRs). A detailed structural analysis led the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR. </p
Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors
In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.</p
Fixing Flavins:Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor
Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.</p
Asymmetric synthesis of optically pure aliphatic amines with an engineered robust ω-transaminase
The production of chiral amines by transaminase-catalyzed amination of ketones is an important application of biocatalysis in synthetic chemistry. It requires transaminases that show high enantioselectivity in asymmetric conversion of the ketone precursors. A robust derivative of ω-transaminase from Pseudomonas jessenii (PjTA-R6) that naturally acts on aliphatic substrates was constructed previously by our group. Here, we explore the catalytic potential of this thermostable enzyme for the synthesis of optically pure aliphatic amines and compare it to the well-studied transaminases from Vibrio fluvialis (Vf TA) and Chromobacterium violaceum (CvTA). The product yields indicated improved performance of PjTA-R6 over the other transaminases, and in most cases, the optical purity of the produced amine was above 99% enantiomeric excess (e.e.). Structural analysis revealed that the substrate binding poses were influenced and restricted by the switching arginine and that this accounted for differences in substrate specificities. Rosetta docking calculations with external aldimine structures showed a correlation between docking scores and synthetic yields. The results show that PjTA-R6 is a promising biocatalyst for the asymmetric synthesis of aliphatic amines with a product spectrum that can be explained by its structural features
A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.Fil: Partipilo, Michele. University of Groningen; PaĂses BajosFil: Yang, Guang. University of Groningen; PaĂses BajosFil: Mascotti, MarĂa Laura. University of Groningen; PaĂses Bajos. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones BiolĂłgicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias FĂsico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones BiolĂłgicas de San Luis; ArgentinaFil: Wijma, Hein J.. University of Groningen; PaĂses BajosFil: Slotboom, Dirk Jan. University of Groningen; PaĂses BajosFil: Fraaije, Marco Wilhelmus. University of Groningen; PaĂses Bajo
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