Hartwig, J.F.

Nature 2016, 534, 534-537, 10.1038/nature17968

Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system1. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations2,3. However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites1,4,5. The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein6,7,8. This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes9,10. In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.

Hartwig, J.F.

Science 2016, 354, 102-106, 10.1126/science.aah4427

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C–H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours−1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C–H bonds and intermolecular carbene insertions into C–H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.

Hartwig, J.F.

ACS Cent. Sci. 2017, 3, 302-308, 10.1021/acscentsci.6b00391

Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C–H bond. We postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, >70:1 dr, >75% yield, and ∼10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Together, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and substrate scope of transition metal catalysts with the exquisite selectivity of enzymes, generating catalysts that enable reactions to occur with levels and modes of activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone.

Hartwig, J.F.

J. Am. Chem. Soc. 2017, 139, 1750-1753, 10.1021/jacs.6b11410

Cytochrome P450 enzymes have been engineered to catalyze abiological C–H bond amination reactions, but the yields of these reactions have been limited by low chemoselectivity for the amination of C–H bonds over competing reduction of the azide substrate to a sulfonamide. Here we report that P450s derived from a thermophilic organism and containing an iridium porphyrin cofactor (Ir(Me)-PIX) in place of the heme catalyze enantioselective intramolecular C−H bond amination reactions of sulfonyl azides. These reactions occur with chemoselectivity for insertion of the nitrene units into C−H bonds over reduction of the azides to the sulfonamides that is higher and with substrate scope that is broader than those of enzymes containing iron porphyrins. The products from C−H amination are formed in up to 98% yield and ∼300 TON. In one case, the enantiomeric excess reaches 95:5 er, and the reactions can occur with divergent site selectivity. The chemoselectivity for C–H bond amination is greater than 20:1 in all cases. Variants of the Ir(Me)-PIX CYP119 displaying these properties were identified rapidly by evaluating CYP119 mutants containing Ir(Me)-PIX in cell lysates, rather than as purified enzymes. This study sets the stage to discover suitable enzymes to catalyze challenging C–H amination reactions.

Brustad, E.M.

ChemBioChem 2017, 18, 2380-2384, 10.1002/cbic.201700397

Engineering an (Ir)regular cytochrome P450: Mutations within the heme‐binding pocket of a cytochrome P450 enabled the selective incorporation of an artificial Ir‐porphyrin cofactor into the protein, in cells. This orthogonal metalloprotein showed enhanced behavior in unnatural carbene‐mediated cyclopropanation of aliphatic and electron‐deficient olefins.

Clark, D.S.; Hartwig, J.F.; Keasling, J.D.; Mukhopadhyay, A.

Nat. Chem. 2021, 13, 1186-1191, 10.1038/s41557-021-00801-3

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.