C–H bonds are ubiquitous structural units of organic molecules; while these bonds are generally considered to be chemically inert, the recent emergence of methods for C–H functionalization promises to transform the way synthetic chemistry is performed. The intermolecular amination of C–H bonds represents a particularly desirable and challenging transformation for which no efficient, highly selective, and renewable catalysts exist. Here we report the directed evolution of an iron-containing enzymatic catalyst, based on a cytochrome P450 monooxygenase, for the highly enantioselective, intermolecular amination of benzylic C–H bonds. The biocatalyst is capable of up to 1,300 turnovers, exhibits excellent enantioselectivities, and provides access to valuable benzylic amines. Iron complexes are generally poor catalysts for C–H amination: in this catalyst, the enzyme’s protein framework confers activity on an otherwise unreactive iron-heme cofactor.

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.protein engineering | allostery | noncanonical amino acid | PLP

Supporting Table 1. NADP-to-NAD cofactor specificity reversal. Structures followed by (h) are homology models, while those followed by another PDB accession code use the cofactor from that protein and (m) denotes a structure of a mutant protein. Mutations indicated in italics are distal to the 2' position. For citation information, see Supporting Material 2.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a caseby-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.branched-chain amino acid pathway | cofactor imbalance K etol-acid reductoisomerases (KARI; EC 1.1.1.86) are a family of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductases that catalyze an alkyl-migration followed by a ketol-acid reduction of (S)-2-acetolactate (S2AL) and 2-aceto-2-hydroxybutyrate to yield (R)-2,3-dihydroxy-isovalerate and (R)-2,3-dihydroxy-3-methylvalerate, respectively (1), which are essential intermediates in the biosynthesis of branched-chain amino acids (BCAAs) (2, 3). The demand for these essential amino acids, used in the preparation of animal feed, human dietary supplements, and pharmaceuticals, is currently estimated to exceed 1,500 tons per year (4). In addition, the BCAA pathway has been engineered to produce fine chemicals and biofuels, including 1-butanol and isobutanol (5, 6). Under the anaerobic conditions preferred for large-scale fermentations, biosynthesis of BCAAs and other products that use this pathway is limited by the pathway's cofactor imbalance and reduced cellular production of NADPH (7,8). One approach to overcoming the cofactor imbalance is to engineer KARI to use NADH generated in glycolysis, thereby enabling anaerobic production of BCAA pathway products (7,8).Efforts to switch the cofactor specificity of oxidoreductases from NADPH to NADH have been made with varying degrees of success (8-17). The three reports of cofactor-switched KARIs (7,8,15) from two different organisms show few commonalities in terms of residues targeted for engineering. A general recipe for switching KARI cofactor specificity would allow metabolic engineers to take advantage of the natural sequence diversity of the KARI family, with concomitant diversity in properties such as expression level, pH tolerance, or thermal stability. By combining a systematic analysis of all reviewed and manually annotated [SwissProt (18)] KARIs, information from our previous work on switching the cofactor specificity of the Escherichia coli KARI (7), and available KARI structures, we have identified a subset of residues in ...