R.B. Leveson-Gower

Science, Volume 384, Issue 6691, Page 106-112, April 2024.

Radical-type carbene transfer catalysis is an efficient method for the direct functionalization of C–H and C=C bonds. However, carbene radical complexes are currently formed via high-energy carbene precursors, such as diazo compounds or iodonium ylides. Many of these carbene precursors require additional synthetic steps, have an explosive nature or generate halogenated waste. Con-sequently, the utilization of carbene radical catalysis is limited by specific carbene precursors to access the carbene radical inter-mediate. In this study, we generate a cobalt(III) carbene radical complex from dimethyl malonate, which is commercially available and bench-stable. EPR and NMR spectroscopy were used to identify the intermediates and showed that the cobalt(III) carbene radical complex is formed upon light irradiation. In presence of styrene, carbene transfer occurred, forming cyclopropane as the product. With this photochemical method, we demonstrate that dimethyl malonate can be used as an alternative carbene precursor in the formation of a cobalt(III) carbene radical complex.

Nature, Published online: 27 March 2024; doi:10.1038/d41586-024-00958-0

Posting about a paper on X seems to boost engagement but not citations. Plus, researchers pinpoint humans’ first home outside Africa and what the science says about the Baltimore bridge collapse.

Nature, Published online: 28 March 2024; doi:10.1038/s41586-024-07341-z

Copper-catalyzed dehydrogenation or lactonization of C(sp³)−H bonds

Due to the scarcity of C–F bond forming enzymatic activities in nature and the contrasting ubiquity of organofluorine moieties in bioactive compounds, developing new biocatalytic fluorination reactions represents a preeminent challenge in enzymology, biocatalysis, and synthetic biology. Additionally, catalytic asymmetric C(sp3)–H fluorination remains a challenging problem facing synthetic chemists. Although many nonheme Fe halogenases have been discovered to promote C(sp3)–H halogenation reactions, to date, efforts to convert these Fe halogenases to fluorinases have remained unsuccessful. We repurposed a plant-derived natural nonheme enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) to catalyze unnatural enantioselective C(sp3)–H fluorination via a radical rebound mechanism. Directed evolution afforded C–H fluorinating enzyme ACCOCHF displaying 200-fold higher activity, substantially improved chemoselectivity and excellent enantioselectivity, converting a range of substrates into enantioenriched organofluorine products. Notably, almost all the beneficial mutations were found to be distal to the Fe centre, underscoring the importance of substrate tunnel engineering in nonheme Fe biocatalysis. Computational studies revealed that the radical rebound step with the Fe(III)–F intermediate has an exceedingly low activation barrier of 3.4 kcal/mol, highlighting a new avenue to expand the catalytic repertoire of enzymes to encompass asymmetric C–F bond formation.

The importance of protein templates in artificial enzyme design is illustrated through genetic code expansion. Incorporation of a secondary amine into the nucleotide-binding DHFR and multidrug-binding LmrR resulted in catalytic entities, with the former favoring the use of NADPH as the hydride source for reactions, whereas the latter required biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH).

Abstract

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Nature Synthesis, Published online: 28 March 2024; doi:10.1038/s44160-024-00514-8

Non-canonical amino acids are important building blocks in the synthesis of natural products, peptides and drugs. Now, a one-pot chemoenzymatic approach to synthesize branched azacyclic non-canonical amino acids is reported. This method combines enzymatic transamination of 2,n-diketoacids and stereocontrolled chemical reduction to provide the desired products with high stereoselectivity.

Nature Synthesis, Published online: 28 March 2024; doi:10.1038/s44160-024-00507-7

Methods for enzymatic C–F bond formation are rare. Now an enzymatic method for enantioselective C(sp3)–F bond formation is reported, through reprogramming non-haem iron enzyme (S)-2-hydroxypropylphosphonate epoxidase. Mechanistic studies reveal that the process proceeds through an iron-mediated radical fluorine transfer process.

Proteins and enzymes can be repurposed by the introduction of artificial cofactors or non-canonical amino acids (ncAAs). These artificial biocatalytic constructs turned into valuable tools to perform new-to-nature reactions with biocatalysts increasing their scope. This perspective focuses on the limitations and future application for in vivo biosynthetic pathways.

Abstract

Astonishing progress has been achieved in unlocking new-to-nature biocatalysis in the past decades. The progress in protein engineering enabled research to efficiently incorporate artificial structural elements into enzyme design. Recent trends include cofactor mimetics, artificial metalloenzymes and non-canonical amino acids. In this perspective article, we present the state-of-the-art, discuss recent examples and our view on what we call artificial biocatalysis. Although these artificial systems undoubtedly increase the scope of biocatalysis, their applicability remains challenging. Fundamental questions regarding the impact of this research field are addressed in this perspective.

Nature Chemistry, Published online: 26 March 2024; doi:10.1038/s41557-024-01500-5

Negatively charged lysine acylations—malonylation, succinylation and glutarylation—impact protein structure and function, which can affect cellular processes. Now temporarily masked thioester derivatives of succinylation and glutarylation can be used for site-specific modification of diverse bacterial and mammalian proteins, which can facilitate the study of how these lysine modifications impact enzymatic activity and control protein–protein and protein–DNA interactions.

Nature Communications, Published online: 21 March 2024; doi:10.1038/s41467-024-46960-y

Prostaglandins are of interest to synthetic chemists due to their biological activities. Here, the authors present a concise chemoenzymatic synthesis method for several representative prostaglandins, achieved in 5 to 7 steps, via the common intermediate bromohydrin, a radical equivalent of Corey lactone.

Science, Volume 383, Issue 6689, Page 1350-1357, March 2024.

Science, Volume 383, Issue 6689, Page 1312-1317, March 2024.

Science, Volume 383, Issue 6689, March 2024.

Metagenomics generally involves isolation and extraction of DNA from various environmental samples, which can then be cloned and expressed into proteins of interest to address difficult chemical reactions in biocatalysis.

Abstract

In the ever-growing demand for sustainable ways to produce high-value small molecules, biocatalysis has come to the forefront of greener routes to these chemicals. As such, the need to constantly find and optimise suitable biocatalysts for specific transformations has never been greater. Metagenome mining has been shown to rapidly expand the toolkit of promiscuous enzymes needed for new transformations, without requiring protein engineering steps. If protein engineering is needed, the metagenomic candidate can often provide a better starting point for engineering than a previously discovered enzyme on the open database or from literature, for instance. In this review, we highlight where metagenomics has made substantial impact on the area of biocatalysis in recent years. We review the discovery of enzymes in previously unexplored or ‘hidden’ sequence space, leading to the characterisation of enzymes with enhanced properties that originate from natural selection pressures in native environments.

Photoenzymatic catalysis is an emerging platform for asymmetric synthesis. In most of these reactions, the protein templates a charge transfer complex between the cofactor and substrate, which absorbs in the blue region of the electromagnetic spectrum. Here, we report the engineering of a photoenzymatic ‘ene’-reductase to utilize red light (620 nm) for a radical cyclization reaction. Mechanistic studies indicate that red light ac-tivity is achieved by introducing a broadly absorbing shoulder off the previously identified cyan absorption feature. Molecular dynamics simulations, docking, and excited-state calculations suggest that red light absorption is a 𝜋→ 𝜋* transition from flavin to the substrate, while the cyan feature is the red-shift of the flavin 𝜋→ 𝜋* transition, which occurs upon substrate binding. Differences in the excitation event help to disfavor alkylation of the flavin cofactor, a pathway for catalyst decomposition observed with cyan light but not red.

Organohalogen compounds are extensively used as building blocks, intermediates, pharmaceuticals, and agrochemicals due to their unique chemical and biological properties. Installing halogen substituents, however, frequently requires functionalized starting materials and multistep functional group interconversion. Several classes of halogenases evolved in nature to enable halogenation of a different classes of substrates; for example, site-selective halogenation of electron rich aromatic compounds is catalyzed by flavin-dependent halogenases (FDHs). Mechanistic studies have shown that these enzymes use FADH2 supplied by a flavin reductase (FRed) to reduce O2 to water with concomitant oxidation of X- to HOX (X = Cl, Br, I). This species travels through a tunnel within the enzyme to access the FDH active site. Here, it is believed to H-bond to an active site lysine proximal to bound substrate, enabling electrophilic halogenation with selectivity imparted via molecular recognition, rather than directing groups or strong electronic activation. The unique selectivity of FDHs led to several early biocatalysis efforts, preparative halogenation was rare, and the hallmark catalyst-controlled selectivity of FDHs did not translate to non-native substrates. FDH engineering was limited to site-directed mutagenesis, which resulted in modest changes in site-selectivity or substrate preference. To address these limitations, we optimized expression conditions for the FDH RebH and its cognate FRed, RebF. We then showed that RebH could be used for preparative halogenation of non-native substrates with catalyst-controlled selectivity. We reported the first examples in which the stability, substrate scope, and site selectivity of an FDH were improved to synthetically useful levels via directed evolution. X-ray crystal structures of evolved FDHs and reversion mutations showed that random mutations throughout the RebH structure were critical to achieving high levels of activity and selectivity on diverse aromatic substrates, and these data were used in combination with molecular dynamics simulations to develop predictive model for FDH selectivity. Finally, we used family-wide genome mining to identify a diverse set of FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds. The diversity of our evolved and mined FDHs allowed us to pursue synthetic applications beyond simple aromatic halogenation. For example, we established that FDHs catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. These results highlight the ability of FDH active sites to tolerate different substrate topologies. This utility was further expanded by our recent studies on the single component FDH/FRed, AetF. While we were initially drawn to AetF because it does not require a separate FRed, we found that it halogenates substrates that are not halogenated efficiently or at all by other FDHs and provides high enantioselectivity for reactions that could only be achieved using RebH variants after extensive mutagenesis. Perhaps most notably, AetF catalyzes site-selective aromatic iodination and enantioselective iodoetherification. Together, these studies highlight the origins of FDH engineering, the utility and limitations of the enzymes developed to date, and the promise of FDHs for an ever-expanding range of biocatalytic halogenation reactions.

Efficient enzyme engineering generates transferases with orders of magnitude higher activity for various substrates in just one round of engineering. The engineered enzymes are highly stereoselective and enable SAM analog (re)generation with iodoalkanes. The enzymes and methods reported might further advance the field of biocatalytic alkylation chemistry with “off-the-shelf” alkylating reagents.

Abstract

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from “off-the-shelf” iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M⁻¹ s⁻¹, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with “off-the-shelf” reagents.

Proceedings of the National Academy of Sciences, Volume 121, Issue 11, March 2024.

Enzymes are molecular machines optimized by nature to allow otherwise impossible chemical processes to occur. Their design is a challenging task due to the complexity of the protein space and the intricate relationships between sequence, structure, and function. Recently, large language models (LLMs) have emerged as powerful tools for modeling and analyzing biological sequences, but their application to protein design is limited by the high cardinality of the protein space. This study introduces a framework that combines LLMs with genetic algorithms (GAs) to optimize enzymes. LLMs are trained on a large dataset of protein sequences to learn relationships between amino acid residues linked to structure and function. This knowledge is then leveraged by GAs to efficiently search for sequences with improved catalytic performance. We focused on two optmization tasks: improving the feasibility of biochemical reactions and increasing their turnover rate. Systematic evaluations on 105 biocatalytic reactions demonstrated that the LLM-GA framework generated mutants outperforming the wild-type enzymes in terms of feasibility in 90% of the instances. Further in-depth evaluation of seven reactions reveals the power of this methodology to make `the best of both worlds' and create mutants with structural features and flexibility comparable to the wild types. Our approach advances the state-of-the-art computational design of biocatalysts, ultimately opening opportunities for more sustainable chemical processes.

Halohydrin dehalogenases (HHDHs) are powerful enzymes for the asymmetric diversification of oxyfunctionalized synthons. They feature two characteristic sequence motifs that distinguish them from homologous short-chain dehydrogenases and reductases. Sequence motif 1, carrying a conserved threonine, glycine and a central aromatic residue, lines the nucleophile binding pocket of HHDHs. It could therefore impact nucleophile binding and presumably also activity of the enzymes. However, experimental evidence supporting this theory is largely missing. Herein, we systematically studied the mutability of the three conserved motif 1 residues as well as their resulting impact on enzyme activity, stability and selectivity in two model HHDHs: HheC from Agrobacterium radiobacter AD1 and HheG from Ilumatobacter coccineus. In both HheC and HheG, the conserved threonine and glycine only tolerated mutations to structurally similar amino acids. In contrast, the central aromatic (i.e., phenylalanine or tyrosine) residue of motif 1 demonstrated much higher variability in HheC. Remarkably, some of these variants featured drastically altered activity, stability and selectivity characteristics. For instance, variant HheC F12Y displayed up to 5-fold increased specific activity in various epoxide ring opening and dehalogenation reactions as well as enhanced enantioselectivity (e.g., an E-value of 74 in the azidolysis of epichlorohydrin compared to E = 22 for HheC wild type), while exhibiting also a 10 K higher apparent melting temperature. QM and MD simulations support the experimentally observed activity increase of HheC F12Y and revealed alterations in the hydrogen bonding network within the active site. As such, our results demonstrate that multiple enzyme properties of HHDHs can be altered through targeted mutagenesis of conserved motif 1 residues. In addition, this work illustrates that motif 1 plays vital roles beyond nucleophile binding by impacting solubility and stability properties. These insights advance our understanding of HHDH active sites and will facilitate their future engineering.

Biocatalysis has played a limited role in the early stages of drug discovery. This is often attributed to the limited substrate scope of enzymes not affording access to vast areas of novel chemical space. Here, we have shown a promiscuous nitroreductase enzyme (NR-55) can be used to produce a panel of functionalised anilines from a diverse panel of aryl nitro starting materials. After screening on analytical scale, we show that sixteen substrates could be scaled to 1 mmol scale, with several poly-functional anilines afforded with ease under the standard conditions. The aniline products were also screened for activity against several cell line of interest, with modest activity observed for one compound. This study demonstrates the potential for nitroreductase biocatalysis to provide access to functional fragments under benign conditions.

The ability of α-amanitin to potently inhibit RNA polymerase II (RNAP II) has elicited further research into its use as a novel payload for antibody-drug conjugates. Despite this promise, the de novo synthesis of α-amanitin is still a major chal-lenge, as it possesses an unusual bicyclic octapeptide structure that contains several oxidized amino acids, most notably 4,5-dihydroxy-L-isoleucine. Here, we report a concise chemoenzymatic synthesis of this key amino acid residue, which features two regio- and diastereoselective enzymatic C–H oxidations on L-isoleucine.