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.

Cavity Carbon-Nanopipette Electrodes for Dopamine Detection.

Microelectrodes are typically used for neurotransmitter detection, but nanoelectrodes are not because there is a trade-off between spatial resolution and sensitivity that is dependent on surface area. Cavity carbon-nanopipette electrodes (CNPEs), with tip diameters of a few hundred nanometers, have been developed for nanoscale electrochemistry. Here, we characterize the electrochemical performance of CNPEs with fast-scan cyclic voltammetry (FSCV) for the first time. Dopamine detection using cavity CNPEs, with a depth equivalent to a few radii, is compared with that using open-tube CNPEs, an essentially infinite geometry. Open-tube CNPEs have very slow temporal responses that change over time as the liquid rises in the CNPE. However, a cavity CNPE has a fast temporal response to a bolus of dopamine that is not different from that of a traditional carbon-fiber microelectrode. Cavity CNPEs, with tip diameters of 200-400 nm, have high currents because the small cavity traps and increases the local dopamine concentration. The trapping also leads to an FSCV frequency-independent response and the appearance of cyclization peaks that are normally observed only with large concentrations of dopamine. CNPEs have high dopamine selectivity over ascorbic acid (AA) because of the repulsion of AA by the negative electric field at the holding potential and the irreversible redox reaction. In mouse-brain slices, cavity CNPEs detected exogenously applied dopamine, showing they do not clog in tissue. Thus, cavity CNPEs are promising neurochemical sensors that provide spatial resolution on the scale of hundreds of nanometers, which is useful for small model organisms or for locations near specific cells.

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.

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.

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.

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.