Engineering Dirhodium Artificial Metalloenzymes for Diazo Coupling Cascade Reactions*.

Artificial metalloenzymes (ArMs) are commonly used to control the stereoselectivity of catalytic reactions, but controlling chemoselectivity remains challenging. In this study, we engineer a dirhodium ArM to catalyze diazo cross-coupling to form an alkene that, in a one-pot cascade reaction, is reduced to an alkane with high enantioselectivity (typically >99 % ee) by an alkene reductase. The numerous protein and small molecule components required for the cascade reaction had minimal effect on ArM catalysis. Directed evolution of the ArM led to improved yields and E/Z selectivities for a variety of substrates, which translated to cascade reaction yields. MD simulations of ArM variants were used to understand the structural role of the cofactor on ArM conformational dynamics. These results highlight the ability of ArMs to control both catalyst stereoselectivity and chemoselectivity to enable reactions in complex media that would otherwise lead to undesired side reactions.

A High-Throughput Method for Directed Evolution of NAD(P)+-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues.

Engineering flavin-free NAD(P)+-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD+s) is of importance for eliminating the need for costly NAD(P)+ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD+ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD+-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD+-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD+-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN+). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency (k cat/K M) and a specific activity of 17.7 U/mg on NMN+, which is comparable to the wild-type enzyme on its natural coenzyme, NADP+. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD+-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNAD+s similar to the levels of natural enzyme/coenzyme pairs.

Iron(II) complexes of dimethyltriazacyclophane.

Treatment of the ortho-triazacyclophane 1,4-dimethyltribenzo[b,e,h][1,4,7]triazacyclonona-2,5,8-triene [(C6H5)3(NH)(NCH3)2, L1] with Fe[N(SiMe3)2]2 yields the dimeric iron(II) complex bis(µ-1,4-dimethyltribenzo[b,e,h][1,4,7]triazacyclonona-2,5,8-trien-7-ido)bis[(µ-1,4-dimethyltribenzo[b,e,h][1,4,7]triazacyclonona-2,5,8-trien-7-ido)iron(II)], [Fe(C20H18N3)4] or Fe2(L1)4 (9). Dissolution of 9 in tetrahydrofuran (THF) results in solvation by two THF ligands and the formation of a simpler monoiron complex, namely bis(µ-1,4-dimethyltribenzo[b,e,h][1,4,7]triazacyclonona-2,5,8-trien-7-ido-κN7)bis(tetrahydrofuran-κO)iron(II), [Fe(C20H18N3)2(C4H8O)2] or (L1)2Fe(THF)2 (10). The reaction is reversible and 10 reverts in vacuo to diiron complex 9. In the structures of both 9 and 10, the monoanionic triazacyclophane ligand L1- is observed in only the less-symmetric saddle conformation. No bowl-shaped crown conformers are observed in the solid state, thus preventing chelating κ3-coordination to the metal as had been proposed earlier based on density functional theory (DFT) calculations. Instead, the L1- ligands are bound in either a η2-chelating fashion through the amide and one amine donor (for one of the four ligands of 9), or solely through their amide N atoms in an even simpler monodentate η1-coordination mode. Density functional calculations on dimer 9 revealed nearly full cationic charges on each Fe atom and no bonding interaction between the two metal centers, consistent with the relatively long Fe...Fe distance of 2.912 (1) Šobserved in the solid state.

Evolving artificial metalloenzymes via random mutagenesis.

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

Selective C-H bond functionalization using repurposed or artificial metalloenzymes.

Catalytic CH bond functionalization has become an important tool for organic synthesis. Metalloenzymes offer a solution to one of the foremost challenges in this field, site-selective CH functionalization, but they are only capable of catalyzing a subset of the CH functionalization reactions known to small molecule catalysts. To overcome this limitation, metalloenzymes have been repurposed by exploiting the reactivity of their native cofactors toward substrates not found in nature. Additionally, new reactivity has been accessed by incorporating synthetic metal cofactors into protein scaffolds to form artificial metalloenzymes. The selectivity and activity of these catalysts has been tuned using directed evolution. This review covers the recent progress in developing and optimizing both repurposed and artificial metalloenzymes as catalysts for selective CH bond functionalization.