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. Please click Additional Files below to see the full abstract

Catalytic mechanism and protein engineering of copper-containing nitrite reductase

The thesis contains work on the catalytic mechanism, and then especially on the reversibility of the reaction and the order of substrate addition and reduction of the catalytically active type-2 copper site. Furthermore, protein engineering of the type-1 copper site is reported and a study into the reorganizational energy of this site.UBL - phd migration 201

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

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 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. Please click Additional Files below to see the full abstract

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

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