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

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

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (kcat) than the original enzyme, but in most cases KM values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening

Nitrofurantoin for the treatment of uncomplicated urinary tract infection in female patients:the impact of dosing regimen, age, and renal function on drug exposure

Purpose: The aim of this study is to determine nitrofurantoin exposure in female patients with different age and renal function with complaints of an uncomplicated UTI. Also the nitrofurantoin exposure in relation to the dosage regimen will be studied. Methods: Eight general practitioners (GP) participated in the study and included 38 patients with symptoms of an uncomplicated UTI, treated either with a dose of 50 mg q6h or 100 mg q12h, upon the discretion of the GP. Nitrofurantoin exposure was quantified in the patient’s 24-h urine samples by UHPLC-UV and the area under the curve was calculated. Results: The 38 patients provided a range of 2–17 urine samples. The urine nitrofurantoin exposure was 1028 mg h/L for the patients receiving 50 mg q6h and 1036 mg h/L for those treated with 100 mg q12h (p = 0.97) and was not affected by age and eGFR (p = 0.64 and p = 0.34, respectively). Conclusion: The data obtained do not support the discouragement of nitrofurantoin use in the elderly and in patients with impaired renal function. Since only a small number of patients were included, a larger study with more patients is warranted to evaluate nitrofurantoin exposure and adverse effects.</p

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence‐related to other ATP‐dependent enzymes involved in lactam hydrolysis, like 5‐oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP‐dependent acetone‐ and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His‐tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6‐aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG(0)'= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)(2) tetramer and revealed an ATP‐binding site in CapA and a Zn‐coordinating site in CapB. Mutations in the ATP‐binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild‐type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP‐dependent lactam hydrolysis

Bioinformatics and Computationally Supported Redesign of Aspartase for β-Alanine Synthesis by Acrylic Acid Hydroamination

Aspartate ammonia lyases catalyze the reversible amination of fumarate to l-aspartate. Recent studies demonstrate that the thermostable enzyme from Bacillus sp. YM55-1 (AspB) can be engineered for the enantioselective production of substituted β-amino acids. This reaction would be attractive for the conversion of acrylic acid to β-alanine, which is an important building block for the preparation of bioactive compounds. Here we describe a bioinformatics and computational approach aimed at introducing the β-alanine synthesis activity. Three strategies were used: First, we redesigned the α-carboxylate binding pocket of AspB to introduce activity with the acrylic acid. Next, different template enzymes were identified by genome mining, equipped with a redesigned α-carboxylate pocket, and investigated for β-alanine synthesis, which yielded variants with better activity. Third, interactions of the SS-loop that covers the active site and harbors a catalytic serine were computationally redesigned using energy calculations to stabilize reactive conformations and thereby further increase the desired β-alanine synthesis activity. Different improved enzymes were obtained and the best variants showed k cat values with acrylic acid of at least 0.6-1.5 s -1 with K M values in the high mM range. Since the β-alanine production of wild-type enzyme was below the detection limit, this suggests that the k cat/ K m was improved by at least 1000-fold. Crystal structures of the 6-fold mutant of redesigned AspB and the similarly engineered aspartase from Caenibacillus caldisaponilyticus revealed that their ligand-free structures have the SS-loop in a closed (reactive) conformation, which for wild-type AspB is only observed in the substrate-bound enzyme. AlphaFold-generated models suggest that other aspartase variants redesigned for acrylic acid hydroamination also prefer a 3D structure with the loop in a closed conformation. The combination of binding pocket redesign, genome mining, and enhanced active-site loop closure thus created effective β-alanine synthesizing variants of aspartase. </p