A Fungal Ascorbate Oxidase with Unexpected Laccase Activity.

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.

Evolving the Promiscuity of Elizabethkingia meningoseptica Oleate Hydratase for the Regio- and Stereoselective Hydration of Oleic Acid Derivatives.

The addition of water to non-activated carbon-carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio- and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D-structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18-fold improved conversion of OA derivatives, while retaining the excellent regio- and stereoselectivity in the olefin hydration reaction.

Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica.

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. © 2017 Wiley Periodicals, Inc.

Exploring the Catalytic Promiscuity of Phenolic Acid Decarboxylases: Asymmetric, 1,6-Conjugate Addition of Nucleophiles Across 4-Hydroxystyrene.

The catalytic promiscuity of a ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) and phenolic acid decarboxylases (PADs) for the asymmetric conjugate addition of water across the C=C bond of hydroxystyrenes was extended to the N-, C- and S-nucleophiles methoxyamine, cyanide and propanethiol to furnish the corresponding addition products in up to 91% ee. The products obtained from the biotransformation employing the most suitable enzyme/nucleophile pairs were isolated and characterized after optimizing the reaction conditions. Finally, a mechanistic rationale supported by quantum mechanical calculations for the highly (S)-selective addition of cyanide is proposed.

PpEst is a novel PBAT degrading polyesterase identified by proteomic screening of Pseudomonas pseudoalcaligenes.

A novel esterase, PpEst, that hydrolyses the co-aromatic-aliphatic polyester poly(1,4-butylene adipate-co-terephthalate) (PBAT) was identified by proteomic screening of the Pseudomonas pseudoalcaligenes secretome. PpEst was induced by the presence of PBAT in the growth media and had predicted arylesterase (EC 3.1.1.2) activity. PpEst showed polyesterase activity on both whole and milled PBAT film releasing terephthalic acid and 4-(4-hydroxybutoxycarbonyl)benzoic acid while end product inhibition by 4-(4-hydroxybutoxycarbonyl)benzoic acid was observed. Modelling of an aromatic polyester mimicking oligomer into the PpEst active site indicated that the binding pocket could be big enough to accommodate large polymers. This is the first report of a PBAT degrading enzyme being identified by proteomic screening and shows that this approach can contribute to the discovery of new polymer hydrolysing enzymes. Moreover, these results indicate that arylesterases could be an interesting enzyme class for identifications of polyesterases.

Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase.

The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.

Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.

An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/ß-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

Structure-Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

Regioselective Enzymatic ß-Carboxylation of para-Hydroxy- styrene Derivatives Catalyzed by Phenolic Acid Decarboxylases.

We report on a 'green' method for the utilization of carbon dioxide as C1 unit for the regioselective synthesis of (E)-cinnamic acids via regioselective enzymatic carboxylation of para-hydroxystyrenes. Phenolic acid decarboxylases from bacterial sources catalyzed the ß-carboxylation of para-hydroxystyrene derivatives with excellent regio- and (E/Z)-stereoselectivity by exclusively acting at the ß-carbon atom of the C=C side chain to furnish the corresponding (E)-cinnamic acid derivatives in up to 40% conversion at the expense of bicarbonate as carbon dioxide source. Studies on the substrate scope of this strategy are presented and a catalytic mechanism is proposed based on molecular modelling studies supported by mutagenesis of amino acid residues in the active site.

CavitOmiX Drug Discovery: Engineering Antivirals with Enhanced Spectrum and Reduced Side Effects for Arboviral Diseases.

Advancing climate change increases the risk of future infectious disease outbreaks, particularly of zoonotic diseases, by affecting the abundance and spread of viral vectors. Concerningly, there are currently no approved drugs for some relevant diseases, such as the arboviral diseases chikungunya, dengue or zika. The development of novel inhibitors takes 10-15 years to reach the market and faces critical challenges in preclinical and clinical trials, with approximately 30% of trials failing due to side effects. As an early response to emerging infectious diseases, CavitOmiX allows for a rapid computational screening of databases containing 3D point-clouds representing binding sites of approved drugs to identify candidates for off-label use. This process, known as drug repurposing, reduces the time and cost of regulatory approval. Here, we present potential approved drug candidates for off-label use, targeting the ADP-ribose binding site of Alphavirus chikungunya non-structural protein 3. Additionally, we demonstrate a novel in silico drug design approach, considering potential side effects at the earliest stages of drug development. We use a genetic algorithm to iteratively refine potential inhibitors for (i) reduced off-target activity and (ii) improved binding to different viral variants or across related viral species, to provide broad-spectrum and safe antivirals for the future.

Folding the human proteome using BioNeMo: A fused dataset of structural models for machine learning purposes.

Human proteins are crucial players in both health and disease. Understanding their molecular landscape is a central topic in biological research. Here, we present an extensive dataset of predicted protein structures for 42,042 distinct human proteins, including splicing variants, derived from the UniProt reference proteome UP000005640. To ensure high quality and comparability, the dataset was generated by combining state-of-the-art modeling-tools AlphaFold 2, OpenFold, and ESMFold, provided within NVIDIA's BioNeMo platform, as well as homology modeling using Innophore's CavitomiX platform. Our dataset is offered in both unedited and edited formats for diverse research requirements. The unedited version contains structures as generated by the different prediction methods, whereas the edited version contains refinements, including a dataset of structures without low prediction-confidence regions and structures in complex with predicted ligands based on homologs in the PDB. We are confident that this dataset represents the most comprehensive collection of human protein structures available today, facilitating diverse applications such as structure-based drug design and the prediction of protein function and interactions.

Targeting the substrate binding site of E. coli nitrile reductase QueF by modeling, substrate and enzyme engineering.

Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase.

Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis.

Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis.

Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis.

A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ µM (native enzyme) to 0.21 and 0.33 s(-1)/µM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

AI-assisted structural consensus-proteome prediction of human monkeypox viruses isolated within a year after the 2022 multi-country outbreak.

IMPORTANCE: The 2022 outbreak of the monkeypox virus already involves, by April 2023, 110 countries with 86,956 confirmed cases and 119 deaths. Understanding an emerging disease on a molecular level is essential to study infection processes and eventually guide drug discovery at an early stage. To support this, we provide the so far most comprehensive structural proteome of the monkeypox virus, which includes 210 structural models, each computed with three state-of-the-art structure prediction methods. Instead of building on a single-genome sequence, we generated our models from a consensus of 3,713 high-quality genome sequences sampled from patients within 1 year of the outbreak. Therefore, we present an average structural proteome of the currently isolated viruses, including mutational analyses with a special focus on drug-binding sites. Continuing dynamic mutation monitoring within the structural proteome presented here is essential to timely predict possible physiological changes in the evolving virus.

Optimizing variant-specific therapeutic SARS-CoV-2 decoys using deep-learning-guided molecular dynamics simulations.

Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of multiple spike RBD/hACE2 variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W and multi-mutation variants as high-affinity candidates, which we validated in vitro with virus neutralization assays. We evaluated binding affinities of these ACE2 variants with the RBDs of Omicron BA.3, Omicron BA.4/BA.5, and Omicron BA.2.75 in silico. In addition, candidates produced in Nicotiana benthamiana, an expression organism for potential large-scale production, showed a 4.6-fold reduction in half-maximal inhibitory concentration (IC50) compared with the same variant produced in CHO cells and an almost six-fold IC50 reduction compared with wild-type hACE2-Fc.

Crystallization of the novel S-adenosyl-L-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis.

Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-L-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, ß = 96.93°.

Crystallization of a novel metal-containing cupin from Acidobacterium sp. and preliminary diffraction data analysis.

Recombinant AciX9_0562 from Acidobacterium sp. MP5ACTX9 (UniProt ID E8WYN5) containing sequence motifs characteristic of the RmlC-type cupins superfamily and containing Pfam motif PF07883 has been successfully cloned, expressed and purified. AciX9_0562 crystallized in a number of conditions from the Morpheus protein crystallization screen. The best crystal diffracted to 2.7 Å resolution (space group C222(1); unit-cell parameters a = 125.29, b = 254.63, c = 82.99 Å). Structure solution was facilitated by the automated molecular-replacement pipeline BALBES. The initial solution was automatically rebuilt using the PHENIX AutoBuild wizard, with final R and R(free) values of 0.23 and 0.26, respectively. The structure is currently undergoing manual refinement.

Recent changes in the mutational dynamics of the SARS-CoV-2 main protease substantiate the danger of emerging resistance to antiviral drugs.

Introduction: The current coronavirus pandemic is being combated worldwide by nontherapeutic measures and massive vaccination programs. Nevertheless, therapeutic options such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main-protease (Mpro) inhibitors are essential due to the ongoing evolution toward escape from natural or induced immunity. While antiviral strategies are vulnerable to the effects of viral mutation, the relatively conserved Mpro makes an attractive drug target: Nirmatrelvir, an antiviral targeting its active site, has been authorized for conditional or emergency use in several countries since December 2021, and a number of other inhibitors are under clinical evaluation. We analyzed recent SARS-CoV-2 genomic data, since early detection of potential resistances supports a timely counteraction in drug development and deployment, and discovered accelerated mutational dynamics of Mpro since early December 2021. Methods: We performed a comparative analysis of 10.5 million SARS-CoV-2 genome sequences available by June 2022 at GISAID to the NCBI reference genome sequence NC_045512.2. Amino-acid exchanges within high-quality regions in 69,878 unique Mpro sequences were identified and time- and in-depth sequence analyses including a structural representation of mutational dynamics were performed using in-house software. Results: The analysis showed a significant recent event of mutational dynamics in Mpro. We report a remarkable increase in mutational variability in an eight-residue long consecutive region (R188-G195) near the active site since December 2021. Discussion: The increased mutational variability in close proximity to an antiviral-drug binding site as described herein may suggest the onset of the development of antiviral resistance. This emerging diversity urgently needs to be further monitored and considered in ongoing drug development and lead optimization.