Vitamin B 12 derivatives catalyze a wide range of organic transformations, but B 12 -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 β-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 β-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 B 12 -dependent enzymes as catalysts for non-native transformations.

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.

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....

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.

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 FADH 2 , 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. Siteselective 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.Flavin-dependent halogenases (FDHs) natively catalyze site-selective halogenation of electron rich aromatic and enolate groups in a diverse range of halogenated natural products. [1][2][3] This unique capability has led to extensive efforts to understand FDH mechanism and the origins of their site selectivity. [4,5] Early studies established that FDH catalysis initially mirrors flavoprotein monooxygenase catalysis in that an enzyme-bound, reduced flavin adenine dinucleotide (FADH 2 ) cofactor reacts with O 2 to generate a hydroperoxy flavin intermediate. [6] In FDHs, this intermediate reacts with bound halide, typically bromide or chloride, to generate HOX, which migrates through the enzyme to a substrate binding pocket. [7][8][9] Most evidence now suggests that hydrogen bonding by a key active site lysine residue activates HOX for electrophilic halogenation, and precise substrate binding leads to site-selective halogenation by this species. [5,10,11] Nearly all FDHs reported to date require a separate flavin reductase to supply FADH 2 , [6,12] and this enzyme is typically driven by a glucose/glucose dehydrogenase cofactor regeneration system for biocatalysis applications (Figure 1A). [13] The need for a separate flavin reductase complicates biocatalysis since these enzymes are not widely available and are typically produced in-house, they add to the protein waste that must be removed during product isolation, and they can lead to undesired background reactions. [14] Previously, our group demonstrated that genetically fusing the flavin reductase RebF to the FDH RebH improved halogenation yields from whole-cell biocatalysis, suggesting that increased local concentration of FADH 2 can improve the efficiency of biocatalysis relative to the free enzymes (Figure 1A). [15] A recent family-wide sequence/

The design of allosteric regulation in proteins to dynamically control function is a challenge in synthetic biology. To address this challenge, we developed an integrated computational and experimental workflow to incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues were genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination would bias the conformations of these enzymes, leading to reversible control of activity. MD-simulations guided rational BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enabled rapid prototyping of variants. Ultimately, this strategy yielded enzymes with a robust 20-fold dynamic range in response to divalent metals 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.

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...

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 the FADH2¬ required by these enzymes. This requirement complicates biocatalysis applications since flavin reductases are not widely available, they add to the protein waste that must be removed during product isolation, and they can lead to undesired background reactions. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates. High site selectivity was observed in many cases, and activity on relatively unactivated substrates and heterocyclic substrates was demonstrated. High enantioselectivity was observed for atroposelective halogenation and halocyclization reactions. Site-selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF, along with the fact that this enzyme requires only a commercially available glucose dehydrogenase to drive its halogenase activity, suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.