R.B. Leveson-Gower

The Front Cover illustrates the design of artificial enzymes incorporating secondary amines-harbouring non-canonical amino acids (ncAAs). The amino acids, referred to as ′life building blocks,′ are depicted as toy building blocks, with canonical amino acids represented by blue and green blocks, while highlighted orange blocks symbolize novel and functional ncAAs. In their Research Article, I. Drienovská and co-workers designed and synthesized a panel of new functional ncAAs. Using the Stop Codon Suppression methodology, D/L-pyrrolidine and D/L-piperidine ncAAs were successfully incorporated into proteins. As a model study, LmrR was utilized for the design of artificial enzymes. The resulting proteins, incorporating D/L-pyrrolidine moieties, exhibited catalytic activity in a model reaction — Michael addition reaction. The incorporation of pyrrolidine- and piperidine-based ncAAs significantly broadens the available toolbox for protein engineering and chemical biology applications. The cover picture prepared by Laura Canil. More information can be found in the Research Article by I. Drienovská and co-workers.

Stereoselectivity-tailored, photoenzyme-catalyzed kinetic resolution of oxalates and oxamic acids prepared from the corresponding sec-alcohols or amines was possible by using a pair of stereocomplementary CvFAP variants with excellent selectivity (both giving up to 99 % e.e., E>200). The enzymes were obtained by focused rational iterative site-specific mutagenesis (FRISM)-guided stereodivergent protein engineering of fatty acid photodecarboxylase.

Abstract

Stereodivergent engineering of one enzyme to create stereocomplementary variants for synthesizing optically pure molecules with tailor-made (R) or (S) configurations on an optional basis is highly desirable and challenging. This study aimed to engineer fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) using the focused rational iterative site-specific mutagenesis (FRISM) strategy to obtain two highly stereocomplementary variants with excellent selectivity (both giving products with up to 99 % e.e.). These variants were used for the CvFAP-catalyzed light-driven kinetic resolution of oxalates or oxamic acids prepared from the corresponding sec-alcohols or amines, providing a new biotransformation process for preparing chiral sec-alcohols and amines. Molecular dynamics simulation, kinetic data and transient spectra revealed the source of selectivity. This study represents the first example of the kinetic resolution of sec-alcohols or amines catalyzed by a pair of stereocomplementary CvFAPs.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond oxidations and allylic hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-b- methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along the evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity towards allylic oxidation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

Nature, Published online: 23 January 2024; doi:10.1038/d41586-024-00165-x

Flavour variations are mainly the result of changes at the chromosome level, sequencing effort finds.

Biocompatible chemistry enables abiotic reactions to be interfaced with metabolic pathways in living microorganisms. This includes both native and de novo biosynthetic processes to access abiotic feedstocks, intermediates and products in vivo. Herein we report a biocompatible Lossen rearrangement that is catalysed by phosphate in the bacterium Escherichia coli for the chemical transformation of activated acyl hydroxamates to primary amines in living cells. Using a para-carboxylated substrate, the biocompatible reaction can be used to generate the metabolite para-aminobenzoic acid (PABA) to rescue ∆pabA/B and ∆aroC auxotrophy and enable cell growth. The Lossen substrate can also be synthesised from polyethylene terephthalate (PET) and applied to whole-cell biocatalytic reactions and fermentations generating industrial small molecule products – including the analgesic and antipyretic drug paracetamol – paving the way for a general strategy to addict E. coli and other industrial chassis strains to PET plastic waste as a bioremediation strategy and for the upcycling of plastic waste using engineered biology. Together, this work showcases how non-enzymatic biocompatible reactions can be interfaced and cooperate with microbial metabolism to expand the available toolbox of metabolic chemistry for small molecule synthesis in native and engineered cellular systems.

Despite the development of numerous advanced ligands for Pd-catalyzed Suzuki cross-coupling reactions, the potential of (oligo)peptides serving as ligands has remained largely unexplored. This study demonstrates, via DFT modeling, that (oligo)peptide ligands can exhibit superior activity compared to classic phosphines in these reactions. The utilization of natural amino acids such as Met, SeMet, and His offers strong binding of the Pd center, thereby ensuring substantial stability of the system. The increasing sustainability and economic viability of (oligo)peptide synthesis open new prospects for applying Pd-(oligo)peptide systems as greener catalysts. The feasibility of de novo engineering an artificial Pd-based enzyme for Suzuki cross-coupling is discussed, laying the groundwork for future innovations in catalytic systems.

Nature, Published online: 10 January 2024; doi:10.1038/s41586-023-06897-6

tRNA display enables the direct selection of orthogonal aminoacyl-tRNA synthetases that acylate orthogonal tRNAs with non-canonical monomers, enabling in vivo synthesis of proteins that include these monomers and expanding the repertoire of the genetic code.

Stereoselective hydrophosphination of alkenes is a promising route to chiral phosphorus compounds, but the use of alkenyl-heteroarenes is challenged by their low reactivity and control of stereoselectivity. Here we present a general Mn(I)-catalysed enantioselective hydrophosphination of alkenyl aza-heteroarenes. The method was applied to a wide range of alkenyl heterocycles and provided access to a non-symmetric P,N,P ligand structure.

Abstract

This paper presents a Mn(I)-catalysed methodology for the enantioselective hydrophosphination of terminal alkenyl aza-heteroarenes. The catalyst operates through H−P bond activation, enabling successful hydrophosphination of a diverse range of alkenyl-heteroarenes with high enantioselectivity. The presented protocol addresses the inherently low reactivity and the commonly encountered suboptimal enantioselectivities of these challenging substrates. As an important application we show that this method facilitates the synthesis of a non-symmetric tridentate P,N,P-containing ligand like structure in just two synthetic steps using a single catalytic system.

The application of biochemical methods in organosilicon chemistry has the potential to provide access to otherwise unattainable chemical structures. The development of biochemical methods in silicon chemistry has been under exploration for approximately 40 years, and recently, methods have been developed for catalyzing transformations at silicon in which proteins have been shown to catalyze new-to-nature transformations. These new methods complement previous work in which enzymes performed more traditional transformations in which silicon generally played the role of spectator. This overview will cover recent developments in the field of organosilicon biotechnology focusing on the use of peptides and proteins to mediate transformations at silicon.

We report the efficient and site selective modification of non-canonical dehydroamino acids in ribosomally synthesized and post-transationally modified peptides (RiPPs) by β-amination. The singly modified thiopeptide Thiostrepton showed an up to 35-fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity was maintained.

Nature, Published online: 18 December 2023; doi:10.1038/s41586-023-06822-x

Enzyme-bound ketyl radicals derived from thiamine diphosphate are selectively generated through single-electron oxidation by a photoexcited organic dye and shown to lead to enantioselective radical acylation reactions.

Nature Catalysis, Published online: 18 December 2023; doi:10.1038/s41929-023-01065-5

Merging photoredox and biocatalysis provides opportunities to address challenges in synthetic chemistry. Now the combination of a ruthenium photocatalyst for oxidative radical formation and ‘ene’-reductases for radical interception enables an enantiodivergent decarboxylative alkylation reaction.

Intermolecular functionalization of tertiary C–H bonds to construct fully substituted stereogenic carbon centers represents a formidable challenge: without the assistance of directing groups, the state-of-the-art catalysts struggle to introduce chirality to racemic tertiary sp3-carbon centers. Direct asymmetric functionalization of such centers is a worthy reactivity and selectivity goal for modern biocatalysis. Here we present an engineered nitrene transferase (P411-TEA-5274), derived from a bacterial cytochrome P450, that is capable of aminating tertiary C–H bonds to provide chiral α-tertiary primary amines with high efficiency (up to 2300 total turnovers) and selectivity (up to >99% enantiomeric excess (e.e.)). The construction of fully substituted stereocenters with methyl and ethyl groups underscores the enzyme’s remarkable selectivity. A comprehensive substrate scope study demonstrates the biocatalyst’s compatibility with diverse functional groups and tertiary C–H bonds. Mechanistic studies, incorporating both experimental and computational data, elucidate how active-site residues distinguish between the enantiomers and enable the enzyme to perform this transformation with excellent enantioselectivity.

The side-chain carboxylates of Asp1 and Asp7 in laspartomycin coordinate with a calcium cation to adopt the active conformation. When these residues were replaced by serine, calcium dependence was eliminated and the resulting synthetic analogue depended instead on phenylboronic acid for its antimicrobial activity.

Abstract

The prevalence of drug-resistant bacterial pathogens foreshadows a healthcare crisis. Calcium-dependent antibiotics (CDAs) are promising candidates to combat infectious diseases as many of them show modes of action (MOA) orthogonal to widespread resistance mechanisms. The calcium dependence is nonetheless one of the hurdles toward realizing their full potential. Using laspartomycin C (LspC) as a model, we explored the possibility of reducing, or even eliminating, its calcium dependence. We report herein a synthetic LspC analogue (B1) whose activity no longer depends on calcium and is instead induced by phenylboronic acid (PBA). In LspC, Asp1 and Asp7 coordinate to calcium to anchor it in the active conformation; these residues are replaced by serine in B1 and condense with PBA to form a boronic ester with the same anchoring effect. Using thin-layer chromatography, MS, NMR, and complementation assays, we demonstrate that B1 inhibits bacterial growth via the same MOA as LspC, i.e., sequestering the cell wall biosynthetic intermediate undecaprenyl phosphate. B1 is as potent and effective as LspC against several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Our success in converting a CDA to a boron-dependent antibiotic opens a new avenue in the design and functional control of drug molecules.

Chemically-driven reaction cycles are prevalent in nature, yet artificial examples are still rare and often lack robustness or versatility. In this study, we introduce acylphosphate steady states that can be accessed from a wide range of organophosphates using either carboxylic anhydride or carbodiimide fuels. The combination of carboxylic anhydride fuel and pyridine catalysis makes this chemistry sufficiently robust to allow for 25 fueling cycles without generation of observable quantities of detrimental side products such as pyrophosphates. We demonstrate that the acylation of organophosphates gives rise to transient aggregates, and we harness the transient fluorescence of acylphosphate-bridged excimers in rapid screenings of more than 50 catalysts in a single well plate experiment. Due to its versatility and robustness, we anticipate that the organophosphate / acylphosphate reaction cycle will prove useful for the creation of chemically-driven molecular machines and transient self-assemblies.

Nature Communications, Published online: 11 December 2023; doi:10.1038/s41467-023-44082-5

Will protein structure search tools like AlphaFold replace protein sequence search with BLAST? We discuss the promises, using structure search for remote homology detection, and why protein BLAST, as the leading sequence search tool, should strive to incorporate structural information

The carbene transfer reaction was realized in the biosynthetic pathway. Using L-limonene as the precursor, L-carveol was biotransformed by P411-PFA to generate unnatural terpenoid derivatives with trifluoromethyl group.

Abstract

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature‘s repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.