R.B. Leveson-Gower

Nature, Published online: 06 March 2024; doi:10.1038/s41586-024-07140-6

Global profiling of hyper-reactive tryptophan sites across whole proteomes using tryptophan chemical ligation by cyclization (Trp-CLiC) reveals a systematic map of tryptophan residues that participate in cation–π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

Vicinal diones and α-keto esters are demonstrated to be electrophiles of both natural and computationally designed aldolases with various catalytic mechanisms, facilitating efficient asymmetric synthesis of small molecules with tertiary alcohols.

Abstract

Aldolases are powerful C−C bond-forming enzymes with high stereoselectivity and broad substrate scope in biocatalysis, but their ability to stereoselectively construct tertiary alcohols has not been fully explored. Herein, we demonstrate that vicinal diones and α-keto esters are electrophiles that can be accepted by both natural and computationally designed aldolases via various catalytic mechanisms. This method allows for the efficient asymmetric synthesis of small molecules with tertiary alcohols, including noncanonical amino acids. This study presents the first types of generic nonnatural substrates of aldolases and reveals new opportunities for the use of aldolases in the synthesis of versatile chiral synthons.

Nature Communications, Published online: 04 March 2024; doi:10.1038/s41467-024-46123-z

The authors previously showed that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita Baylis-Hillman (MBH) reaction. Here, they report another efficient MBHase that employs a non-canonical Nδ-methylhistidine nucleophile paired with a catalytic glutamate, providing an alternative mechanistic solution for MBH catalysis.

Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (Nε methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and five phosphorylations were successfully prepared and isolated in high yield and purity. Our methodology was then applied in the production of ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thereby enables the systematic study of PTM function at a molecular level, and offers a convenient way for an expedited screening of constructs.

To unravel why computational design fails in creating viable enzymes, while directed evolution (DE) succeeds, our research delves into the laboratory evolution of Protoglobin. DE has adapted this protein to efficiently catalyze carbene transfer reactions. We show that the previously proposed enhanced substrate access and binding alone cannot account for increased yields during DE. The 3D electric field in the entire active site is tracked through protein dynamics, clustered using the affinity propagation algorithm, and subjected to principal component analysis. This analysis reveals notable changes in the electric field with DE, where distinct field topologies influence transition state energetics and mechanism. A chemically meaningful field component emerges and takes the lead during DE and facilitates crossing the barrier to carbene transfer. Our findings underscore intrinsic electric field dynamic's influence on enzyme function, the ability of the field to switch mechanisms within the same protein, and the crucial role of the field in enzyme design.

Cerium photoredox catalysis has emerged as a powerful strategy to activate molecules under mild conditions. Radical intermediates are formed using visible light and simple complexes of the earth-abundant lanthanide. However, it remains a major challenge to achieve stereocontrol in these reactions. Here, we report an artificial photoenzyme enabling this chemistry inside a protein. We utilize a de novo designed protein scaffold that tightly binds lanthanide ions in its central cavity. Upon visible-light irradiation, the cerium-dependent enzyme catalyzes the radical C–C bond cleavage of 1,2-diols in aqueous solution. Protein engineering led to variants with improved photostability and initial stereoselectivity. The photoenzyme cleaves a range of aromatic and aliphatic substrates, including lignin surrogates. Surface display of the protein scaffold on E. coli facilitates whole-cell photobiocatalysis. Furthermore, we show that also natural lanthanide-binding proteins are suitable for this approach. Our study thus demonstrates a new-to-nature enzymatic photoredox activity with broad catalytic potential.

Nature Chemistry, Published online: 05 March 2024; doi:10.1038/s41557-024-01468-2

The rapid generation of molecular complexity from a given molecular scaffold is crucial to drug discovery and development. Now the chemodivergent molecular editing of indoles using fluoroalkyl carbenes has been developed to modularly access four different types of fluorine-containing N-heterocyclic compound with high molecular complexity.

CarH photoreceptors bound to photolabile cobalt corrin coenzyme B₁₂ (AdoCbl) are DNA-binding tetramers that repress genes for carotenoid synthesis in the dark. Light triggers AdoCbl photolysis and CarH tetramer disassembly to activate gene expression. AdoRhbl, a synthetic photostable isostructural rhodium surrogate of AdoCbl, emulates AdoCbl in CarH binding, tetramerization and DNA binding yet blocks photoregulation by CarH in vitro and in vivo.

Abstract

Coenzyme B₁₂ (AdoCbl; 5′-deoxy-5′-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound “base-off/His-on” to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B₁₂, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Nature Chemistry, Published online: 28 February 2024; doi:10.1038/s41557-024-01463-7

Developing a generalizable method for blocking and rescuing tryptophan (Trp) interactions would enable the gain-of-function manipulation of various Trp-containing proteins but has so far been challenging. Now a genetically encoded N1-vinyl-caged Trp capable of rapid and bioorthogonal decaging enables site-specific activation of Trp on a protein of interest within living cells.

Nature Catalysis, Published online: 23 February 2024; doi:10.1038/s41929-024-01117-4

The reasons for epistasis, wherein mutations interact non-additively, are often not fully understood. Now it is found that shifting the rate-limiting step from substrate binding to the chemical reaction step during the directed evolution of β-lactamase correlates with epistasis.

The integration of organocatalysis and enzyme catalysis in one-pot cascade processes allows for the efficient construction of complex molecular architectures with high levels of stereocontrol. However, challenges related to reaction compatibility between both processes are often a limitation for the development of efficient synthetic routes. In this study, we describe the combination of an enzymatic aerobic oxidation followed by the squaramide-mediated asymmetric formation of C-P and C-C bonds to access important building blocks such as chiral α-hydroxy phosphonates and β-nitro alcohols in good yields and enantiomeric ratios. This sequential process is conducted in a one-pot fashion within a biphasic system and represents a pioneering example of a chemoenzymatic cascade involving aerobic biooxidation and an organocatalytic step operating under hydrogen-bond activation mode.

Multiple enzymes catalyze the formation of pyrroloindoles from indoles, usually coupled with a functional group transfer in the 3-position. In this work, two high-throughput complementary absorbance-based assays were developed for the monitoring of substrate depletion (indole) and product formation (pyrroloindole). The assays were used successfully for enzymatic activity determination, but can be also used for the quantification of natural products.

Abstract

Indoles and pyrroloindoles are structural motifs present in many biologically active natural products. Multiple classes of enzymes catalyze the transformation of indoles into pyrroloindoles via group transfer followed by intramolecular cyclization, such as peroxydases, methyltransferases, and prenyltransferases. Due to the selective introduction of a stereogenic center, these enzymes receive increasing attention as catalytic tools for the production of pharmacologically relevant compounds. Two new colorimetric assays are described in this work, which allow for the quantification of such enzymatic reactions from the perspective of the substrate and the product. For the substrates, the indole assay is based on a modified version of the Ehrlich test, with the use of light as a driving force for color formation. The pyrroloindole assay uses cerium sulfate as a reagent for the colorimetric quantification of the enzymatic products. The assays are complementary and both were successfully utilized for enzymatic activity determination of a C3-indole methyltransferase. They can facilitate high-throughput screening of mutant libraries, offering support for the engineering of such enzymes, but can also be used as stand-alone methods for the detection and quantification of natural products.

The Co(II) analogue of the designed peptide Zn(II)(TRIL23H)₃ is made as a spectroscopic probe for the carbonic anhydrase activity exhibited by this de novo designed system. The coordination geometry of Co(II) and Zn(II) are vastly different at high and low pH. Nonetheless, the Co(II) analogue at high pH exceeds the catalytic efficiency of the parent Zn(II) protein.

Abstract

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes’ first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d⁷ electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H₂O/OH⁻)]_N(TRIL2WL23H)₃ ⁿ⁺ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH⁻)]_N(TRIL2WL23H)₃ ⁺ peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pK _a for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(N_imid)₃(OH)(H₂O) species. The pH dependent kinetic analysis indicates the maximal rate (k_cat), and thus the catalytic efficiency (k_cat/K_m), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

Relying on ubiquitous alkenes, carboamination reactions enable the difunctionalization of the double bond by the concurrent formation of a C–N and a C–C single bond. In the past years, several groups have reported on elegant strategies for the carboamination of alkenes relying on homogeneous catalysts or enzymes. Herein, we report on an artificial metalloenzyme for the enantioselective carboamination of dihydrofuran. Genetic optimization, combined with a Bayesian optimization of catalytic performance, afforded the disubstituted tetrahydrofuran product in up to 22 TON and 85% ee. X-ray analysis of the evolved artificial carboaminase shed light on critical amino acid residues that affect catalytic performance.

Nature, Published online: 14 February 2024; doi:10.1038/s41586-024-07032-9

Ultrafast time-resolved serial femtosecond crystallography is used to investigate a photodissociation reaction in a protein, revealing the strong impact of the pump laser fluence on the structural changes and the reaction mechanism.

An enzyme and a peptide catalyze—in an aqueous buffer—a two-step cascade reaction with high chemo- and stereoselectivity in one pot. The optimization of the modular peptide catalyst and the identification of common reaction conditions were key for bringing the two worlds of enzyme and peptide catalysis together.

Abstract

Enzymes and peptide catalysts consist of the same building blocks but require vastly different environments to operate best. Herein, we show that an enzyme and a peptide catalyst can work together in a single reaction vessel to catalyze a two-step cascade reaction with high chemo- and stereoselectivity. Abundant linear alcohols, nitroolefins, an alcohol oxidase, and a tripeptide catalyst provided chiral γ-nitroaldehydes in aqueous buffer. High yields (up to 92 %) and stereoselectivities (up to 98 % ee) were achieved for the cascade through the rational design of the peptide catalyst and the identification of common reaction conditions.

Although ethers are common in secondary natural products, they are an underrepresented functional group in primary metabolism. As such, there are comparably few enzymes capable of constructing ether bonds in a general fashion. However, such enzymes are highly sought after for synthetic applications as they typically operate with higher regioselectivity and under milder conditions than traditional organochemical approaches. To expand the repertoire of well characterized ether synthases, we herein report on a promiscuous archaeal prenyltransferase from the scarcely researched family of geranylgeranylglyceryl phosphate synthases (GGGPSs or G3PSs). We show that the ultrastable Archaeoglobus fulgidus G3PS makes various (E)- and (Z)-configured prenyl glycerol ethers from the corresponding pyrophosphates, while exerting perfect control over the configuration at the glycerol unit. Based on experimental and computational data, we propose a mechanism for this enzyme which involves an intermediary prenyl carbocation equivalent. As such, this study provides the fundamental understanding and methods to introduce G3PSs into the biocatalytic alkylation toolbox.