R.B. Leveson-Gower

The biomimetic regeneration system for S-adenosylmethionine (SAM) and SAM analogues presented is based on the salvage of the adenine moiety and in situ supply of d-ribose and polyphosphate. It is compatible with a broad range of SAM-dependent enzymes including aminopropyl transferases, and is shown to support ethylation reactions with both conventional and radical SAM methyltransferases.

Abstract

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Engineered biocatalysis: Here we show that the scope of reductive aminases (RedAms) can be extended to allow selective reductive aminations of cyclic secondary amines, such as piperidines and morpholines, with both aldehydes and ketones. These biotransformations provide access to important motifs found in active pharmaceutical ingredients and other bioactive molecules.

Abstract

Reductive aminases (RedAms) have recently emerged as promising biocatalysts for the synthesis of chiral secondary amines by coupling primary amines with aldehydes/ketones. However, access to tertiary amines remains more problematic, particularly when coupling ketones with secondary amines. Here we show that the scope of these enzymes can be extended to allow selective reductive aminations of cyclic secondary amines, such as piperidines and morpholines, with both aldehydes and ketones. These biotransformations provide access to important motifs found in active pharmaceutical ingredients and other bioactive molecules. RedAm-361, discovered from a metagenomic library, was engineered via directed evolution to allow efficient coupling of cyclic amines with carbonyl partners, including dynamic kinetic resolutions of α-functionalized aldehydes and enantioselective amination of ketones. These RedAms now serve as valuable scaffolds for the engineering of industrial biocatalysts to produce key pharmaceutical intermediates.

Anaerobic flow biocatalysis: An engineered cytochrome c enzyme, capable of carbon-silicon bond formation, was immobilised for the first time using the SpyTag/SpyCatcher bioconjugation system. When used in a continuous flow reactor, the immobilised enzyme was able to produce organosilicons with up to 6-fold increased turnover numbers compared to the free enzyme in conventional batch experiments.

Abstract

The recent development of tailored cytochrome enzymes has enabled “new-to-nature” reactivities, such as the biocatalytic formation of carbon-silicon bonds using the cytochrome c from Rhodothermus marinus. To maximise the potential of this remarkable biocatalyst by increasing its turnover numbers (TON) and to enable its reusability in continuous processes, we report the use of the SpyTag/SpyCatcher bioconjugation system to immobilise this enzyme. We successfully modified the enzyme with a SpyTag without significant effects on its catalytic activity. Even after immobilization on microparticles the enzyme retained 60 % activity. When the immobilized enzyme was used in sequential batch or continuous flow to produce an organosilicon, we observed up to 6-fold higher turnover numbers over a total period of 10 days compared to the free enzyme reaction, however we observed a drop in stereoselectivity under these conditions. This is the first report on the successful immobilisation of a carbon-silicon bond forming enzyme for the continuous, biocatalytic production of organosilicons.

Proceedings of the National Academy of Sciences, Volume 120, Issue 12, March 2023.

The role of peptides is nowadays relevant in fields such as drug discovery and biotechnology. Computational analyses are required to study their properties and gain insights into rational design strategies. Both natural and modified peptides containing non-natural amino acids require customized tools to run sequence and structure-based studies. PepFun 2.0 is a new version of the python package for the study of natural and modified peptides using a set of modules to analyze the sequence and structure of the molecules. PepFun 2.0 comprises five main modules for different tasks such as sequence alignments, prediction of properties, generation of conformers, modification of structures, detection of protein-peptide interactions, and extra functions to include peptides containing non-natural amino acids. The code and tutorial are available at: https://github.com/rochoa85/PepFun2

Nature, Published online: 20 March 2023; doi:10.1038/s41586-023-05950-8

Enantioconvergent Cu-catalyzed N-alkylation of aliphatic amines

Nature, Published online: 17 March 2023; doi:10.1038/d41586-023-00831-6

Editors threaten to resign over ‘no-reject’ model that others see as the future of research journals.

Germylenes with two ylide-substituents are usually highly nucleophilic species. Herein, we demonstrate that introduction of a cyano moiety into the ylide backbone decreases the LUMO energy and thus significantly enhances the electrophilic nature of these species. This is reflected in the reactivity but also in the molecular structure of the diylidylgermylene.

Abstract

Owing to the strong electron-donating ability of ylide substituents, diylidyltetrylenes are usually highly nucleophilic species with strong donor capacities. Here, we demonstrate that their electronic properties are in fact highly flexible and can be effectively tuned through variation of the substituent in the ylide backbone. Initial density functional theory studies showed that cyano groups are particularly capable in lowering the LUMO energy of diylidyl germylenes thus turning these usually highly nucleophilic species into electrophilic compounds. This was confirmed by experimental studies. Attempts to synthesize the germylene (Y_CN)₂Ge [with Y_CN=Ph₃P-(C)-CN] from the corresponding metalated ylide Y_CNK selectively led to germanide [(Y_CN)₃Ge)K]₂ thus reflecting the electrophilic nature of the intermediate formed germylene. XRD analysis of single crystals of (Y_CN)₂Ge – serendipitously obtained through protonative cleavage of one ylide from the germanide – revealed a monomeric structure with rather long Ge-ylide linkages, which corroborates well with a pure single bond and no stabilization of the empty p_π orbital at germanium through π bonding. The germanide exhibits methanide-like reactivity towards chalcogens but a likewise weak Ge−C bond as demonstrated by the insertion of carbon dioxide.

The conjugation of Au complexes with proteins and enzymes, generating new types of artificial metalloenzymes, has proven to be interesting and effective to obtain materials with improved properties such as higher stability, catalytic activity and selectivity. In this work, a novel method has been developed for the synthesis and design of artificial gold metalloenzymes at 50ºC in aqueous media, using two genetically modified variants of the alkalophilic lipase Geobacillus thermocatenulatus (GTL). The only difference between these two enzymatic variants is the possible coordination of the Au via active site (GTL-114) or Lid site (GTL-193). TEM analysis of the metalloenzymes revealed the formation of Au (0) nanoparticles with different structures (nanowires, nanorods, nanoshells, nanoclusters) and sizes depending on the mutant and the pH used during the synthesis. Characterisation by fluorescence spectroscopy demonstrated that conjugation of the enzyme to Au altered the tertiary structure of the protein. On the other hand, all metalloenzymes showed excellent reductase like activity. Finally, the selectivity of the enzyme-Au bioconjugates was tested in the asymmetric reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. The protein environment played a key role in the reactivity and selectivity of the metalloenzymes, obtaining chiral nanoparticles with an enantiomeric excess of up to 39% towards (R)-1-phenylethanol after two hours of reaction using GTL-114 pH 10 as catalyst.

By using Rosetta enzyme design and MD simulations, the active site of a thermostable ketoreductase was redesigned to invert the stereoselectivity. Simultaneous introduction of 6–8 mutations without laboratory screening of mutant libraries gave active enzymes catalyzing asymmetric synthesis of 1-phenylethanols with high enantiomeric excess.

Abstract

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6–8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of k_cat /K _M values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

We report a homogeneous photocatalytic system based on cobalt porphyrin substituted myoglobin (CoMb) for CO₂ to CO conversion in water. By optimizing the reaction conditions, our catalysts achieved up to 2000 TON(CO) at low enzyme concentrations, and a product selectivity of 80 % with an increased enzyme loading. We show that the efficiencies of CO generation and overall TON(CO) can be improved by introducing positively charged residues near the active site of CoMb.

Abstract

While native CO₂-reducing enzymes display remarkable catalytic efficiency and product selectivity, few artificial biocatalysts have been engineered to allow understanding how the native enzymes work. To address this issue, we report cobalt porphyrin substituted myoglobin (CoMb) as a homogeneous catalyst for photo-driven CO₂ to CO conversion in water. The activity and product selectivity were optimized by varying pH and concentrations of the enzyme and the photosensitizer. Up to 2000 TON(CO) was attained at low enzyme concentrations with low product selectivity (15 %), while a product selectivity of 74 % was reached by increasing the enzyme loading but with a compromised TON(CO). The efficiency of CO generation and overall TON(CO) were further improved by introducing positively charged residues (Lys or Arg) near the active stie of CoMb, which demonstrates the value of tuning the enzyme secondary coordination sphere to enhance the CO₂-reducing performance of a protein-based photocatalytic system.

Nature, Published online: 13 March 2023; doi:10.1038/d41586-023-00761-3

Zoologist Zuofu Xiang’s research in Asia helps governments to protect the populations and teaches him the value of cooperation.

Science Advances, Volume 9, Issue 10, March 2023.

This study significantly expands the scope of multiple noncanonical amino acid (ncAA) mutagenesis in mammalian cells by developing a tryptophanyl-tRNA synthetase/tRNA pair that efficiently decodes the TGA stop codon. It can be combined with three previously established pairs to create new ways to incorporate up to three distinct ncAAs into a protein in mammalian cells. Using this technology, two distinct cytotoxic drugs were site-specifically conjugated to a full-length humanized antibody.

Abstract

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Here, we report the design and application of multi-step enzymatic cascades to synthesize enantioenriched epoxides and vicinal aromatic triols from simple biomass-derived starting materials in one pot. These artificial metabolic pathways involve a tailor-made aldolase, a highly evolved cofactor-independent peroxyzyme, and when needed a specifically chosen epoxide hydrolase. These attractive biocatalytic cascades can be performed under environmentally benign conditions, such as the use of aqueous media and mild temperatures, and do not require the isolation of reaction intermediates. Good to excellent conversions, high enantioselectivity, and moderate to good product yields are achieved.

Abstract

Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.

Nature, Published online: 08 March 2023; doi:10.1038/s41586-023-05781-7

Structural and biochemical studies of the Mycobacterium smegmatis hydrogenase Huc provides insights into how [NiFe] hydrogenases oxidize trace amounts of atmospheric hydrogen and transfer the electrons liberated via quinone transport.