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

Progressive myoclonus epilepsy (PME) is a syndrome characterized by myoclonic seizures (lightning-like jerks), generalized convulsive seizures, and varying degrees of neurological decline, especially ataxia and dementia. Previously, we characterized three pedigrees of individuals with PME and ataxia, where either clinical features or linkage mapping excluded known PME loci. This report identifies a mutation in PRICKLE1 (also known as RILP for REST/NRSF interacting LIM domain protein) in all three of these pedigrees. The identified PRICKLE1 mutation blocks the PRICKLE1 and REST interaction in vitro and disrupts the normal function of PRICKLE1 in an in vivo zebrafish overexpression system. PRICKLE1 is expressed in brain regions implicated in epilepsy and ataxia in mice and humans, and, to our knowledge, is the first molecule in the noncanonical WNT signaling pathway to be directly implicated in human epilepsy.

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable () cytochrome High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in cytochrome has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

We recently demonstrated that variants of cytochrome P450BM3 (CYP102A1) catalyze the insertion of nitrogen species into benzylic C–H bonds to form new C–N bonds. An outstanding challenge in the field of C–H amination is catalyst-controlled regioselectivity. Here, we report two engineered variants of P450BM3 that provide divergent regioselectivity for C–H amination—one favoring amination of benzylic C–H bonds and the other favoring homo-benzylic C–H bonds. The two variants provide nearly identical kinetic isotope effect values (2.8–3.0), suggesting that C–H abstraction is rate-limiting. The 2.66-Å crystal structure of the most active enzyme suggests that the engineered active site can preorganize the substrate for reactivity. We hypothesize that the enzyme controls regioselectivity through localization of a single C–H bond close to the iron nitrenoid.