Jared C. Lewis

Enantioselective Intramolecular C-H Amination Catalyzed by Engineered Cytochrome P450 Enzymes In Vitro and In Vivo

Nitrogen activation: Though P450 enzymes are masters of oxygen activation and insertion into C-H bonds, their ability to use nitrogen for the same purpose has so far not been explored. Engineered variants of cytochrome P450_(BM3) have now been found to catalyze intramolecular C-H aminations in azide substrates. Mutations to two highly conserved residues significantly increased this activity.

Combinatorial alanine substitution enables rapid optimization of cytochrome P450BM3 for selective hydroxylation of large substrates.

Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_(BM3) variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates.

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450BM3) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine.

Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation

Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor–acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium–carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

Regioselective arene halogenation using the FAD-dependent halogenase RebH.

Co-expression of the halogenase RebH with GroEL/ES and fusion of the flavin reductase RebF to MBP enabled production of both enzymes on scales sufficient for preparative regioselective oxidative halogenation of arenes. The activity and selectivity of RebH contrast with those reported for PrnA, a structurally homologous halogenase, which provided a narrower substrate scope and only enabled halogenation of unnatural substrates at their most electronically activated positions.

A General Method for Artificial Metalloenzyme Formation through Strain‐Promoted Azide–Alkyne Cycloaddition

Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both Siuf8ffH and olefin insertion reactions involving these carbene precursors.

Metallopeptide catalysts and artificial metalloenzymes containing unnatural amino acids

Metallopeptide catalysts and artificial metalloenzymes built from peptide scaffolds and catalytically active metal centers possess a number of exciting properties that could be exploited for selective catalysis. Control over metal catalyst secondary coordination spheres, compatibility with library based methods for optimization and evolution, and biocompatibility stand out in this regard. A wide range of unnatural amino acids (UAAs) have been incorporated into peptide and protein scaffolds using several distinct methods, and the resulting UAAs containing scaffolds can be used to create novel hybrid metal-peptide catalysts. Promising levels of selectivity have been demonstrated for several hybrid catalysts, and these provide a strong impetus and important lessons for the design of and optimization of hybrid catalysts.

Directed Evolution of RebH for Site‐Selective Halogenation of Large Biologically Active Molecules

We recently characterized the substrate scope of wild-type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for the site-selective halogenation of biologically active compounds.

Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution.

We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3‐LR, with a Topt over 5u2009°C higher than that of wild‐type, and 3‐LSR, with a Tm 18u2009°C higher than that of wild‐type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non‐natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.

Engineering Flavin-Dependent Halogenases.

In the two decades since the discovery of the first flavin-dependent halogenase (FDH), great strides have been made in elucidating the diversity of enzymes in this class as well as their structures and functions. More recently, efforts to engineer these enzymes for synthetic applications have yielded their first successes. FDH variants with improved stability, expanded substrate scope, and altered regioselectivity have been developed. Here, we review these efforts and provide representative protocols that have been employed for FDH engineering. Random and structure-guided mutagenesis approaches and screening methods, including HPLC, mass spectrometry, and spectrophotometric methods, are discussed. The protocols described herein should be generalizable to many FDHs and a wide variety of engineering goals.