R.B. Leveson-Gower

Taste perception plays a pivotal role in guiding nutrient intake and aiding in the avoidance of potentially harmful substances through five basic tastes - sweet, bitter, umami, salty, and sour. Taste perception originates from molecular interactions in the oral cavity between taste receptors and chemical tastants. Hence, the recognition of taste receptors and the subsequent perception of taste heavily rely on the physicochemical properties of food ingredients. In recent years, several advances have been made towards the development of machine learning-based algorithms to classify chemical compounds' tastes using their molecular structures. Despite the great efforts, there remains significant room for improvement by developing multi-class models to predict the entire spectrum of basic tastes. Here, we present a multi-class predictor aimed at distinguishing three different tastes, i.e., bitter, sweet, and umami, from other taste sensations. The developed model has been integrated into a publicly accessible web platform. This work lays the groundwork for a comprehensive understanding of the molecular features that drive the perception of tastes, paving the way towards new methodologies in the rational design of foods, such as the pre-determination of specific tastes, the engineering of complementary diets to traditional pharmacological treatments, and many others.

Aldaric acids are attractive diacids that can be prepared by selective oxidation of carbohydrates. The discovery, biochemical and structural characterization of a VAO-type flavin-containing carbohydrate oxidase from Citrus sinensis: URAO_Cs3 is reported. The selective oxidation of D-galacturonic acid in a complex mixture is demonstrated.

Abstract

Aldaric acids are attractive diacids that can be prepared by selective oxidation of carbohydrates. For this, effective biocatalysts are in demand. This work reports on the discovery, biochemical and structural characterization of a VAO-type flavin-containing carbohydrate oxidase from Citrus sinensis: URAO_Cs3. URAO_Cs3 could be overexpressed using prokaryotic and eukaryotic expression systems. Extensive biochemical characterization revealed that the enzyme displays a high thermostability and an exquisite selectivity for uronic acids, galacturonic acid and glucuronic acid. The enzyme was further investigated by determining the crystal structure. The selective oxidation of D-galacturonic acid in a complex mixture was demonstrated, showing how URAO_Cs3 was found to be highly effective in selectively producing galactaric acid while leaving other carbohydrates untouched. In addition to the specific discovery of URAO_Cs3, these findings suggest that plant proteomes can be an interesting source for new biocatalysts.

Herein we synthesized a panel of non-canonical amino acids (ncAAs) harboring functional secondary amines inspired by organocatalysts. After their synthesis and characterization, D/L-pyrrolidine- and D/L-piperidine-based ncAAs were successfully site-specifically incorporated into proteins via stop codon suppression methodology. To demonstrate the utility of these ncAAs, the catalytic performance of the obtained artificial enzymes was investigated in a model Michael addition reaction.

Abstract

Enzymes are attractive catalysts for chemical industries, and their use has become a mature alternative to conventional chemical methods. However, biocatalytic approaches are often restricted to metabolic and less complex reactivities, given the limited amount of functional groups present. This drawback can be addressed by incorporating non-canonical amino acids (ncAAs) harboring new-to-nature chemical groups. Inspired by organocatalysis, we report the design, synthesis and characterization of a panel of ncAAs harboring functional secondary amines and their cellular incorporation into different protein scaffolds. D/L-pyrrolidine- and D/L-piperidine-based ncAAs were successfully site-specifically incorporated into proteins via stop codon suppression methodology. To demonstrate the utility of these ncAAs, the catalytic performance of the obtained artificial enzymes was investigated in a model Michael addition reaction. The incorporation of pyrrolidine- and piperidine- based ncAAs significantly expands the available toolbox for protein engineering and chemical biology applications.

Communications Chemistry, Published online: 11 September 2023; doi:10.1038/s42004-023-00998-z

Microbial enzymes are capable of degrading certain synthetic polymers, with most polyethylene terephthalate (PET) degrading enzymes known to derive from bacteria or fungi. Here, the authors describe an archaeal originating feruloyl-esterase PET46 enzyme with a flexible lid domain and PET degradation capability.

Efficient directed evolution protocols for nonribosomal peptide synthetases are needed to adapt the structures of antibiotic peptides for the fight against antimicrobial resistance. Here, an easily reproducible directed evolution protocol was used to reprogram the synthetase for the antibiotic peptide gramicidin S. A few mutations were sufficient to incorporate the non-standard building block piperazic acid instead of proline with perfect specificity.

Abstract

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N−N bond. This success was achieved by UPLC-MS/MS-based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

The β-subunit of tryptophan synthase was engineered for efficient N-alkylation to access densely functionalized non-canonical amino acids. Mechanistic analysis guided preparative scale synthesis, adding a valuable new enzyme to the biocatalytic toolbox.

Abstract

Non-canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,β-diamino acids. In this study, we used directed evolution to engineer the β-subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re-entry into the catalytic cycle and subsequent decomposition. Additional equivalents of l-serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β-substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

Oxygenation and amination reactions are widespread in synthetic chemistry to produce valuable compounds. Nowadays, the importance of sustainable strategies to introduce oxygen and amino functionalities into organic molecules is increasing. This review discusses recent examples of multi-step biocatalytic cascades involving oxy- and amino-functionalization reactions to produce value-added compounds such as pharmaceuticals and polymer precursors.

Abstract

Biocatalytic cascades are a powerful tool for building complex molecules containing oxygen and nitrogen functionalities. Moreover, the combination of multiple enzymes in one pot offers the possibility to minimize downstream processing and waste production. In this review, we illustrate various recent efforts in the development of multi-step syntheses involving C−O and C−N bond-forming enzymes to produce high value-added compounds, such as pharmaceuticals and polymer precursors. Both in vitro and in vivo examples are discussed, revealing the respective advantages and drawbacks. The use of engineered enzymes to boost the cascades outcome is also addressed and current co-substrate and cofactor recycling strategies are presented, highlighting the importance of atom economy. Finally, tools to overcome current challenges for multi-enzymatic oxy- and amino-functionalization reactions are discussed, including flow systems with immobilized biocatalysts and cascades in confined nanomaterials.

This review highlights how engineered enzymes have been developed and implemented to help address environmental challenges. Topics include the use of engineered enzymes for improving carbon capture and utilisation, bioremediation, plastic deconstruction, and renewable feedstock generation. Successes, challenges, and opportunities for future enzyme engineering campaigns to improve environmental sustainability are discussed.

Abstract

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO₂) and have been embedded into new efficient metabolic CO₂ fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Biocatalytic group transfer reactions can efficiently generate highly complex molecules under mild and environmentally friendly conditions. This Minireview highlights inspiring examples of the development of non-natural donor reagents to power such enzymatic transformations, while discussing the design aspects behind the choice of reagent.

Abstract

Biocatalysis has become a major driver in the innovation of preparative chemistry. Enzyme discovery, engineering and computational design have matured to reliable strategies in the development of biocatalytic processes. By comparison, substrate engineering has received much less attention. In this Minireview, we highlight the idea that the design of synthetic reagents may be an equally fruitful and complementary approach to develop novel enzyme-catalysed group transfer chemistry. This Minireview discusses key examples from the literature that illustrate how synthetic substrates can be devised to improve the efficiency, scalability and sustainability, as well as the scope of such reactions. We also provide an opinion as to how this concept might be further developed in the future, aspiring to replicate the evolutionary success story of natural group transfer reagents, such as adenosine triphosphate (ATP) and S-adenosyl methionine (SAM).

Halogenated heteroarenes are key building blocks across numerous chemical industries. Here, we report that vanadium haloperoxidases are capable of producing 3-haloindoles through decarboxylative halogenation of 3-carboxyindoles. This biocatalytic method is applicable to decarboxylative chlorination, bromination, and iodination in moderate to high yields and with excellent chemoselectivity.

Selenium (Se) is an essential micronutrient that is found naturally in proteins, nucleic acids, and natural products. Unlike selenoproteins and selenonucleic acids, little is known about the structures of the biosynthetic enzymes that incorporate Se into small molecules. Here, we report the X-ray crystal structure of SenB, the first known Se-glycosyltransferase that was recently found to be involved in the biosynthesis of the Se-containing metabolite selenoneine. SenB catalyzes C–Se bond formation using selenophosphate and an activated uridine diphosphate sugar as a Se and glycosyl donor, respectively, making it the first known selenosugar synthase and only one of four bona fide C–Se bond-forming enzymes discovered to date. Our crystal structure, determined to 2.25 Å resolution, reveals that SenB is a type B glycosyltransferase, displaying the prototypical fold with two globular Rossmann-like domains and a catalytic interdomain cleft. By employing complementary structural biology techniques, we find that SenB undergoes both local and global substrate-induced conformational changes, demonstrating a significant increase in α-helicity and a transition to a more compact conformation. Our results provide the first structure of SenB and set the stage for further biochemical characterization in the future.

Biosynthetic modifications of the 6/10-bicyclic hydrocarbon skeletons of the eunicellane family of diterpenoids are un-known. We explored the biosynthesis of a bacterial trans-eunicellane natural product, albireticulone A (3), and identified a novel isomerase that catalyzes a cryptic isomerization in the biosynthetic pathway. We also assigned functions of two cyto-chromes P450 that oxidize the eunicellane skeleton, one of which was a naturally evolved non-functional P450 that when genetically repaired, catalyzes allylic oxidation. Finally, we describe the chemical susceptibility of the trans-eunicellane skeleton to undergo Cope rearrangement to yield inseparable atropisomers.

Enzymatic reactions are an ecofriendly, selective and versatile addition, sometimes even alternative to organic reactions for the synthesis of chemical compounds such as pharmaceuticals or fine chemicals. To identify suitable reactions, computational models to predict the activity of enzymes on non-native substrates, to perform retrosynthetic pathway searches, or to predict the outcomes of reactions including regio- and stereoselectivity are becoming increasingly important. However, current approaches are substantially hindered by the limited amount of available data, especially if balanced and atom mapped reactions are needed and if the models feature machine learning components. We therefore constructed a high-quality dataset (EnzymeMap) by developing a large set of correction and validation algorithms for recorded reactions in the literature and showcase its significant positive impact on machine learning models of retrosynthesis, forward prediction, and regioselectivity prediction, outperforming previous approaches by a large margin. Our dataset allows for deep learning models of enzymatic reactions with unprecedented accuracy, and is freely available online.

Despite the unique reactivity of vitamin B12 and its derivatives, B12-dependent enzymes remain underutilized in biocatalysis. In this study, we repurpose the B12-dependent transcription factor CarH to enable non-native radical cyclization reactions. An engineered variant of this enzyme, CarH*, catalyzes the formation γ- and δ-lactams via either redox-neutral or reductive ring closure with marked enhancement of reactivity and selectivity relative to the free B12 cofactor. CarH* also catalyzes an unusual spirocyclization via dearomatization of pendant arenes to produce bicyclic 1,3-diene products instead of 1,4-dienes provided by existing methods. These results and associated mechanistic studies highlight the importance of protein scaffolds for controlling the reactivity of B12 and expanding the synthetic utility of B12-dependent enzymes.

Conformationally flexible tertiary amine—thiourea-urea catalysts for the Mannich reaction between imines and malonate esters are efficiently inhibited by quaternary ammonium halides. NMR titrations, isothermal titration calorimetry and NOE experiments showed that the catalysts bind chloride and bromide ions with relatively high affinities (K = 103–105 M–1 in acetonitrile), and they refold into catalytically inactive conformations upon complexation. At substoichiometric inhibitor:catalyst ratios, the reactions displayed hypersensitivity to the inhibitors, with overall rates that were lower than those expected from simple 1st order kinetics and 1:1 inhibitor:catalyst stoichiometry. The Mannich reactions turned out to be 2nd order in catalyst, and this finding also readily explains the observed hypersensitivity.

Solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) are powerful methods for obtaining peptides and proteins that are otherwise inaccessible. Nonetheless, numerous sequences are difficult to prepare via SPPS, and cleaved peptides often have low solubility. To address these challenges, we developed a “Synthesis Tag” consisting of six arginines connected via cleavable MeDbz linker. “SynTag” effectively improves batch- and flow-SPPS of “difficult sequences”, enhances solubility of the cleaved peptides and provides direct access to native sequences by hydrolysis, or peptide thioesters for NCL.. We demonstrate its utility in the first chemical synthesis of the MYC transactivation domain (143 AA) with a single NCL. We envisage SynTag to become a broadly applicable tool that enables the synthesis and study of previously unattainable peptides and proteins.

Horseradish peroxidase (HRP) is an archetypal heme-containing metalloenzyme that uses peroxide to oxidize various substrates. Capitalizing on a well-established catalytic mechanism, diverse peroxidase mimics have been widely investigated and optimized. Herein, we report on the design, assembly, characterization, and genetic engineering of an artificial heme-based peroxidase relying on the biotin-streptavidin technology. The crystal structure of both the wild-type and the best-performing double mutant of artificial peroxidase provided valuable insight regarding the nearby residues that were strategically mutated to optimize the peroxidase activity (i.e. Sav S112E K121H). We hypothesize that these two residues mimic the two key second coordination residues involved in activating the bound peroxide in HRP (i.e. Arg 38 and His 42). The evolved artificial peroxidase exhibited best-in-class activity for oxidizing two standard substrates (TMB and ABTS) in the presence of hydrogen peroxide.

Nature Chemistry, Published online: 11 September 2023; doi:10.1038/s41557-023-01333-8

Merging carbonyls to form an alkene by removing their oxygens is rare, yet synthetically useful, and the selective combination of two different carbonyls is especially challenging. Now, two strategies for cross-metathesis of unbiased carbonyls have been developed. An Fe-catalysed carbene/ylide strategy affords Z-alkenes, while Cr-catalysed gem-di-metals yield E-alkenes.

Nature, Published online: 06 September 2023; doi:10.1038/s41586-023-06310-2

We report a small-organic-molecule oscillator that catalyses an independent chemical reaction in situ without impairing its oscillating properties, allowing the construction of complex systems enhancing applications in automated synthesis and systems and polymerization chemistry.

Science, Volume 381, Issue 6660, Page 836-836, August 2023.

Science, Volume 381, Issue 6662, Page 1040-1041, September 2023.

Nature Catalysis, Published online: 31 August 2023; doi:10.1038/s41929-023-01018-y

Anthocyanins are used in the food and cosmetic industries. Due to the insufficient production in alternative hosts, they are still isolated from plants. Now, this study suggests an important catalytic role of glutathione transferases for the efficient biosynthesis of these natural products.