Selective C-H Halogenation of Alkenes and Alkynes Using Flavin-Dependent Halogenases.

Single component flavin-dependent halogenases (FDHs) possess both flavin reductase and FDH activity in a single enzyme. We recently reported that the single component FDH AetF catalyzes site-selective bromination and iodination of a variety of aromatic substrates and enantioselective bromolactonization and iodoetherification of styrenes bearing pendant carboxylic acid or alcohol substituents. Given this inherent reactivity and selectivity, we explored the utility of AetF as catalyst for alkene and alkyne C-H halogenation. We find that AetF catalyzes halogenation of a range of 1,1-disubstituted styrenes, often with high stereoselectivity. Despite the utility of haloalkenes for cross-coupling and other applications, accessing these compounds in a stereoselective manner typically requires functional group interconversion processes, and selective halogenation of 1,1'-disubstituted olefins remains rare. We also establish that AetF and homologues of this enzyme can halogenate terminal alkynes. Mutagenesis studies and deuterium kinetic isotope effects are used to support a mechanistic proposal involving covalent catalysis for halogenation of unactivated alkynes by AetF homologues. These findings expand the scope of FDH catalysis and continue to show the unique utility of single component FDHs for biocatalysis.

Non-native Intramolecular Radical Cyclization Catalyzed by a B12 -Dependent Enzyme.

Despite the unique reactivity of vitamin B12 and its derivatives, B12 -dependent enzymes remain underutilized in biocatalysis. In this study, we repurposed 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 through 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 by 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.

Non-Native Site-Selective Enzyme Catalysis.

The ability to site-selectively modify equivalent functional groups in a molecule has the potential to streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary and secondary metabolism, but leveraging this capability for non-native substrates and reactions requires a detailed understanding of the potential and limitations of enzyme catalysis and how these bounds can be extended by protein engineering. In this review, we discuss representative examples of site-selective enzyme catalysis involving functional group manipulation and C-H bond functionalization. We include illustrative examples of native catalysis, but our focus is on cases involving non-native substrates and reactions often using engineered enzymes. We then discuss the use of these enzymes for chemoenzymatic transformations and target-oriented synthesis and conclude with a survey of tools and techniques that could expand the scope of non-native site-selective enzyme catalysis.

Iridium(III) polypyridine artificial metalloenzymes with tunable photophysical properties: a new platform for visible light photocatalysis in aqueous solution.

Artificial metalloenzymes (ArMs) can combine the unique features of both metal complexes and enzymes by incorporating a cofactor within a protein scaffold. Herein, we describe a panel of ArMs constructed by covalently linking Ir(III) polypyridyl complexes into a prolyl oligopeptidase scaffold. Spectroscopic methods were used to examine how properties of the resulting ArMs are influenced by structural variation of the cyclometalated ligands and the protein scaffold. Visible light photocatalysis by these hybrid catalysts was also examined, leading to the finding that they catalyze inter/intra-molecular [2 + 2] photocycloaddition in aqueous solution. Low but reproducible enantioselectivity was observed using a cofactor that undergoes partial kinetic resolution upon bioconjugation within the ArM active site, showing the importance of scaffold/cofactor interactions for enabling selective ArM photocatalysis. Further evidence of the importance of cofactor/scaffold interactions was provided by analyzing native POP peptidase catalysis by the ArMs. Together, these studies show how Ir(III)-based ArMs constitute a promising starting point for ongoing studies to control the stereoselectivity of EnT reactions by engineering substrate binding/activation motifs in POP.

Asymmetric catalysis by flavin-dependent halogenases.

In nature, flavin-dependent halogenases (FDHs) catalyze site-selective chlorination and bromination of aromatic natural products. This ability has led to extensive efforts to engineer FDHs for selective chlorination, bromination, and iodination of electron rich aromatic compounds. On the other hand, FDHs are unique among halogenases and haloperoxidases that exhibit catalyst-controlled site selectivity in that no examples of enantioselective FDH catalysis in natural product biosynthesis have been characterized. Over the past several years, our group has established that FDHs can catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. Achieving high activity and selectivity for these reactions has required extensive mutagenesis and mitigation of problems resulting from hypohalous acid generated during FDH catalysis. The single-component flavin reductase/FDH AetF is unique among the wild type enzyme we have studied in that it provides high activity and selectivity toward several asymmetric transformations. These results highlight the ability of FDH active sites to tolerate different substrate topologies and suggest that they could be useful for a broad range of oxidative halogenations.

First and second sphere interactions accelerate non-native N-alkylation catalysis by the thermostable, methanol-tolerant B12-dependent enzyme MtaC.

The corrinoid protein MtaC, which is natively involved in methyl transferase catalysis, catalyzes N-alkylation of aniline using ethyl diazoacetate. Our results show how the native preference of B12 scaffolds for radical versus polar chemistry translates to non-native catalysis, which could guide selection of B12-dependent proteins for biocatalysis. MtaC also has high thermal stability and organic solvent tolerance, remaining folded even in pure methanol.

Directed Evolution of an Iron(II)- and α-Ketoglutarate-Dependent Dioxygenase for Site-Selective Azidation of Unactivated Aliphatic C-H Bonds.

FeII - and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of C-H bonds via a variety of C-X bond forming reactions, but achieving high chemoselectivity for functionalization using non-native functional groups remains rare. The current study shows that directed evolution can be used to engineer variants of the dioxygenase SadX that address this challenge. Site-selective azidation of succinylated amino acids and a succinylated amine was achieved as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C-H functionalization with other non-native functional groups.

Analysis of Laboratory-Evolved Flavin-Dependent Halogenases Affords a Computational Model for Predicting Halogenase Site Selectivity.

Flavin-dependent halogenases (FDHs) catalyze selective halogenation of electron-rich aromatic compounds without the need for harsh oxidants required by conventional oxidative halogenation reactions. Predictive models for halogenase site selectivity could greatly improve their utility for chemical synthesis. Toward this end, we analyzed the structures and selectivity of three halogenase variants evolved to halogenate tryptamine with orthogonal selectivity. Crystal structures and reversion mutations revealed key residues involved in altering halogenase selectivity. Density functional theory calculations and molecular dynamics simulations are both consistent with hypohalous acid as the active halogenating species in FDH catalysis. This model was used to accurately predict the site selectivity of halogenase variants toward different synthetic substrates, providing a valuable tool for implementing halogenases in biocatalysis efforts.

The Single-Component Flavin Reductase/Flavin-Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins.

Flavin-dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply them with FADH2 , which complicates biocatalysis applications. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates using a commercially available glucose dehydrogenase to drive its halogenase activity. High site selectivity, activity on relatively unactivated substrates, and high enantioselectivity for atroposelective bromination and bromolactonization was demonstrated. Site-selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.

Institutional decarbonization scenarios evaluated against the Paris Agreement 1.5 °C goal.

Scientifically rigorous guidance to policy makers on mitigation options for meeting the Paris Agreement long-term temperature goal requires an evaluation of long-term global-warming implications of greenhouse gas emissions pathways. Here we employ a uniform and transparent methodology to evaluate Paris Agreement compatibility of influential institutional emission scenarios from the grey literature, including those from Shell, BP, and the International Energy Agency. We compare a selection of these scenarios analysed with this methodology to the Integrated Assessment Model scenarios assessed by the Intergovernmental Panel on Climate Change. We harmonize emissions to a consistent base-year and account for all greenhouse gases and aerosol precursor emissions, ensuring a self-consistent comparison of climate variables. An evaluation of peak and end-of-century temperatures is made, with both being relevant to the Paris Agreement goal. Of the scenarios assessed, we find that only the IEA Net Zero 2050 scenario is aligned with the criteria for Paris Agreement consistency employed here. We investigate root causes for misalignment with these criteria based on the underlying energy system transformation.

Directed Evolution of Flavin-Dependent Halogenases for Site- and Atroposelective Halogenation of 3-Aryl-4(3H)-Quinazolinones via Kinetic or Dynamic Kinetic Resolution.

In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site- and atroposelective halogenation of 3-aryl-4(3H)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving the analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the (M)-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translate well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate binding. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation in the literature, these results suggest that flavin-dependent halogenases (FDHs) could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.

Non-Native Anionic Ligand Binding and Reactivity in Engineered Variants of the Fe(II)- and α-Ketoglutarate-Dependent Oxygenase, SadA.

Mononuclear non-heme Fe(II)- and α-ketoglutarate-dependent oxygenases (FeDOs) catalyze a site-selective C-H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to an FeDO facial triad variant, SadX, and the effects of that binding on HO• vs X• rebound. We establish not only that chloride and bromide enable non-native halogenation reactions but also that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3•, NCO•, and OCN• rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand leads us to calculate barriers for HO• and F• rebound from a putative Fe(III)(OH)(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states rather than an inaccessible barrier for F• rebound. Together, these results improve our understanding of the FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving these ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

Cobalamin-Mediated Electrocatalytic Reduction of Ethyl Chloroacetate in Dimethylformamide.

The catalytic reduction of ethyl chloroacetate (ECA) by hydroxocobalamin (HOCbl) in dimethylformamide was studied electrochemically and spectroelectrochemically to identify initial steps in the reaction between the electrogenerated Co(I) center of cobalamin (cob(I)alamin) and ECA. Cyclic voltammograms of HOCbl in the presence of ECA show a small increase in current related to reduction of Co(II) to Co(I), and a new peak at more negative potentials related to reduction of an ethyl carboxymethyl-Cbl intermediate. The oxidation state of HOCbl during catalysis was monitored by means of spectroelectrochemical controlled-potential bulk electrolysis. Addition of ECA to electrogenerated cob(I)alamin initially generates the Co(II) form (cob(II)alamin) followed by a gradual formation of an ethyl carboxymethyl-Cbl intermediate. Controlled-potential bulk electrolysis was performed to identify products formed from catalytic reduction of ECA by electrogenerated cob(I)alamin and quantify the number of electrons transferred per molecule of ECA. Product distributions and coulometric results, together with the results of voltammograms and spectroelectrochemical controlled-potential bulk electrolysis, were interpreted to propose a reaction mechanism.

Metal-responsive regulation of enzyme catalysis using genetically encoded chemical switches.

Dynamic control over protein function is a central challenge in synthetic biology. To address this challenge, we describe the development of an integrated computational and experimental workflow to incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues are genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination biases the conformations of these enzymes, leading to reversible control of activity. Computational analysis and molecular dynamics simulations are used to rationally guide BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enable rapid prototyping of variants. Ultimately, this strategy yields enzymes with a robust 20-fold dynamic range in response to divalent metal salts over 24 on/off switches, demonstrating the potential of this approach. We envision that this strategy of genetically encoding chemical switches into enzymes will complement other protein engineering and synthetic biology efforts, enabling new opportunities for applications where precise regulation of protein function is critical.

Realization of Paris Agreement pledges may limit warming just below 2 °C.

Over the last five years prior to the Glasgow Climate Pact1, 154 Parties have submitted new or updated 2030 mitigation goals in their nationally determined contributions and 76 have put forward longer-term pledges. Quantifications of the pledges before the 2021 United Nations Climate Change Conference (COP26) suggested a less than 50 per cent chance of keeping warming below 2 degrees Celsius2-5. Here we show that warming can be kept just below 2 degrees Celsius if all conditional and unconditional pledges are implemented in full and on time. Peak warming could be limited to 1.9-2.0 degrees Celsius (5%-95% range 1.4-2.8 °C) in the full implementation case-building on a probabilistic characterization of Earth system uncertainties in line with the Working Group I contribution to the Sixth Assessment Report6 of the Intergovernmental Panel on Climate Change (IPCC). We retrospectively project twenty-first-century warming to show how the aggregate level of ambition changed from 2015 to 2021. Our results rely on the extrapolation of time-limited targets beyond 2030 or 2050, characteristics of the IPCC 1.5 °C Special Report (SR1.5) scenario database7 and the full implementation of pledges. More pessimistic assumptions on these factors would lead to higher temperature projections. A second, independent emissions modelling framework projected peak warming of 1.8 degrees Celsius, supporting the finding that realized pledges could limit warming to just below 2 degrees Celsius. Limiting warming not only to 'just below' but to 'well below' 2 degrees Celsius or 1.5 degrees Celsius urgently requires policies and actions to bring about steep emission reductions this decade, aligned with mid-century global net-zero CO2 emissions.

Controlling Non-Native Cobalamin Reactivity and Catalysis in the Transcription Factor CarH.

Vitamin B12 derivatives catalyze a wide range of organic transformations, but B12-dependent enzymes are underutilized in biocatalysis relative to other metalloenzymes. In this study, we engineered a variant of the transcription factor CarH, called CarH*, that catalyzes styrene C-H alkylation with improved yields (2-6.5-fold) and selectivity relative to cobalamin. While the native function of CarH involves transcription regulation via adenosylcobalamin (AdoCbl) Co(III)-carbon bond cleavage and ß-hydride elimination to generate 4',5'-didehydroadenosine, CarH*-catalyzed styrene alkylation proceeds via non-native oxidative addition and olefin addition coupled with a native-like ß-hydride elimination. Mechanistic studies on this reaction echo findings from earlier studies on AdoCbl homolysis to suggest that CarH* selectivity results from its ability to impart a cage effect on radical intermediates. These findings lay the groundwork for the development of B12-dependent enzymes as catalysts for non-native transformations.

Controlling the optical and catalytic properties of artificial metalloenzyme photocatalysts using chemogenetic engineering.

Visible light photocatalysis enables a broad range of organic transformations that proceed via single electron or energy transfer. Metal polypyridyl complexes are among the most commonly employed visible light photocatalysts. The photophysical properties of these complexes have been extensively studied and can be tuned by modifying the substituents on the pyridine ligands. On the other hand, ligand modifications that enable substrate binding to control reaction selectivity remain rare. Given the exquisite control that enzymes exert over electron and energy transfer processes in nature, we envisioned that artificial metalloenzymes (ArMs) created by incorporating Ru(ii) polypyridyl complexes into a suitable protein scaffold could provide a means to control photocatalyst properties. This study describes approaches to create covalent and non-covalent ArMs from a variety of Ru(ii) polypyridyl cofactors and a prolyl oligopeptidase scaffold. A panel of ArMs with enhanced photophysical properties were engineered, and the nature of the scaffold/cofactor interactions in these systems was investigated. These ArMs provided higher yields and rates than Ru(Bpy)3 2+ for the reductive cyclization of dienones and the [2 + 2] photocycloaddition between C-cinnamoyl imidazole and 4-methoxystyrene, suggesting that protein scaffolds could provide a means to improve the efficiency of visible light photocatalysts.

Expanding the Reactivity of Flavin Dependent Halogenases Toward Olefins via Enantioselective Intramolecular Haloetherification and Chemoenzymatic Oxidative Rearrangements.

Of the different classes of halogenases characterized to date, flavin dependent halogenases (FDHs) are most associated with site-selective halogenation of electron rich arenes and enol(ate) moieties in the biosynthesis of halogenated natural products. This capability has made them attractive biocatalysts, and extensive efforts have been devoted to both discovering and engineering these enzymes for different applications. We have established that engineered FDHs can catalyze different enantioselective halogenation processes, including halolactonization of simple alkenes with a tethered carboxylate nucleophile. In this study, we expand the scope of this reaction to include alcohol nucleophiles and a greater diversity of alkene substitution patterns to access a variety of chiral tetrahydrofurans. We also demonstrate that FDHs can be interfaced with ketoreductases to enable halocyclization using ketone substrates in one-pot cascade reactions and that the halocyclization products can undergo subsequent rearrangements to form hydroxylated and halogenated products. Together, these advances expand the utility of FDHs for enantio- and diastereoselective olefin functionalization.

Can updated climate pledges limit warming well below 2°C?

Increased ambition and implementation are essential.

Phage-Assisted Continuous Evolution and Selection of Enzymes for Chemical Synthesis.

Ligand-dependent biosensors are valuable tools for coupling the intracellular concentrations of small molecules to easily detectable readouts such as absorbance, fluorescence, or cell growth. While ligand-dependent biosensors are widely used for monitoring the production of small molecules in engineered cells and for controlling or optimizing biosynthetic pathways, their application to directed evolution for biocatalysts remains underexplored. As a consequence, emerging continuous evolution technologies are rarely applied to biocatalyst evolution. Here, we develop a panel of ligand-dependent biosensors that can detect a range of small molecules. We demonstrate that these biosensors can link enzymatic activity to the production of an essential phage protein to enable biocatalyst-dependent phage-assisted continuous evolution (PACE) and phage-assisted continuous selection (PACS). By combining these phage-based evolution and library selection technologies, we demonstrate that we can evolve enzyme variants with improved and expanded catalytic properties. Finally, we show that the genetic diversity resulting from a highly mutated PACS library is enriched for active enzyme variants with altered substrate scope. These results lay the foundation for using phage-based continuous evolution and selection technologies to engineer biocatalysts with novel substrate scope and reactivity.