The Fox-1 protein regulates alternative splicing of tissuespecific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded b-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U 1 , G 2 , and C 3 are wrapped around a single phenylalanine, while G 2 and A 4 form a base-pair. This novel RNA binding site is independent from the b-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.

Using rational design, an engineered myoglobin-based catalyst capable of catalyzing the cyclopropanation of aryl-substituted olefins with catalytic proficiency (up to 46,800 turnovers) and excellent diastereo- and enantioselectivity (98–99.9%) was developed. This transformation could be carried out in the presence of up to 20 g / L−1 olefin substrate with no loss in diastereo- and/or enantioselectivity. Mutagenesis and mechanistic studies support a cyclopropanation mechanism mediated by an electrophilic, heme-bound carbene species and a model is provided to rationalize the stereopreference of the protein catalyst. This work shows that myoglobin constitutes a promising and robust scaffold for the development of biocatalysts with carbene transfer reactivity.

SummaryThe evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the 'lineage' from a naturally-occurring C 12 -C 20 fatty acid hydroxylase (P450 BM3 ) to a laboratory-evolved P450 propane monooxygenase (P450 PMO ) having 20 heme domain substitutions compared to P450 BM3 . Biochemical, crystallographic and computational analyses show that a minimal perturbation of the P450 BM3 fold and substrate binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (~9,000-fold increase in k cat /K m ) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely re-specialize the cytochrome P450 for function on a non-native substrate.

Engineered variants of the heme-containing protein myoglobin can efficiently catalyze the insertion of α-diazo esters into the N—H bond of arylamines, featuring a combination of high chemoselectivity, elevated turnover numbers, and broad substrate scope.

The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si–H and C–H insertions, Doyle-Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities.

The development of catalytic systems for the controlled oxidation of C–H bonds remains a highly sought-after goal in chemistry owing to the great utility of such transformation toward expediting the synthesis and functionalization of organic molecules. Cytochrome P450 monooxygenases are the catalysts of choice in the biological world for mediating the oxidation of sp3 and sp2 C–H bonds with a high degree of chemo-, regio-, and stereoselectivity and in a wide array of compounds of varying complexity. The efficiency of these enzymes, compared with chemical methods, to catalyze the insertion of oxygen into unactivated C–H bonds under mild reaction conditions has sparked interest among researchers toward investigating and exploiting P450s for a variety of synthetic applications. Realizing the synthetic potential of these enzymes, however, depends upon the availability of effective strategies to tune the reactivity of natural P450s to obtain viable oxidation catalysts for the desired transformation. This review describes recent efforts in this area involving the use of protein engineering, substrate engineering, guest/host activation, and functional screening strategies. The development of engineered P450s for drug metabolite production and emerging methodologies involving the integration of P450-catalyzed transformations in preparative-scale chemoenzymatic syntheses are also presented. Key challenges that need to be addressed to capitalize on P450 oxidation catalysis for chemical synthesis are discussed.

The direct amination of aliphatic C—H bonds represents a most valuable transformation in organic chemistry. While a number of transition metal-based catalysts have been developed and investigated for this purpose, the possibility to execute this transformation with biological catalysts has remained largely unexplored. Here, we report that cytochrome P450 enzymes can serve as efficient catalysts for mediating intramolecular benzylic C—H amination reactions in a variety of arylsulfonyl azide compouds. Under optimized conditions, the P450 catalysts were found to support up to 390 total turnovers leading to the formation of the desired sultam products with excellent regioselectivity. In addition, the chiral environment provided by the enzyme active site allowed for the reaction to proceed in a stereo- and enantioselective manner. The C—H amination activity, substrate profile, and enantio/stereoselectivity of these catalysts could be modulated by utilizing enzyme variants with engineered active sites.

Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclopropanations, but variants featuring predictable, complementary stereoselectivity in these reactions have remained elusive. In this study, a rationally driven strategy was implemented and applied to engineer myoglobin variants capable of providing access to 1-carboxy-2-aryl-cyclopropanes with high trans-(1R,2R) selectivity and catalytic activity. The stereoselectivity of these cyclopropanation biocatalysts complements that of trans-(1S,2S)-selective variants developed here and previously. In combination with whole-cell biotransformations, these stereocomplementary biocatalysts enabled the multigram synthesis of the chiral cyclopropane core of four drugs (Tranylcypromine, Tasimelteon, Ticagrelor, TRPV1 inhibitor 24) in high yield and with excellent diastereo- and enantioselectivity (98–99.9% de; 96–99.9% ee). These biocatalytic strategies outperform currently available methods to produce these drugs.