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Biaryl compounds, with two connected aromatic rings, are found across medicine, materials science and asymmetric catalysis 1 , 2 . The necessity of joining arene building blocks to access these valuable compounds has inspired several approaches for biaryl bond formation and challenged chemists to develop increasingly concise and robust methods for this task 3 . Oxidative coupling of two C–H bonds offers an efficient strategy for the formation of a biaryl C–C bond; however, fundamental challenges remain in controlling the reactivity and selectivity for uniting a given pair of substrates 4 , 5 . Biocatalytic oxidative cross-coupling reactions have the potential to overcome limitations inherent to numerous small-molecule-mediated methods by providing a paradigm with catalyst-controlled selectivity 6 . Here we disclose a strategy for biocatalytic cross-coupling through oxidative C–C bond formation using cytochrome P450 enzymes. We demonstrate the ability to catalyse cross-coupling reactions on a panel of phenolic substrates using natural P450 catalysts. Moreover, we engineer a P450 to possess the desired reactivity, site selectivity and atroposelectivity by transforming a low-yielding, unselective reaction into a highly efficient and selective process. This streamlined method for constructing sterically hindered biaryl bonds provides a programmable platform for assembling molecules with catalyst-controlled reactivity and selectivity. A study presents a biocatalytic method for the formation of sterically hindered biaryl bonds, providing a tunable approach for assembling molecules with catalyst-controlled reactivity, site selectivity and atroposelectivity.

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.Natural products are produced by living organisms practising nature’s chemical transformations. Now, an unnatural product has been generated by creating hybrid biosynthetic microorganisms. These microorganisms combine an unnatural chemical transformation—catalysis by an artificial metalloenzyme containing an iridium-based, unnatural cofactor—with a natural biosynthetic pathway within the same cell.

Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) approximately 100 kilocalories per mole], with an apparent second-order rate constant of kapp = 1.1 x 10⁷ per molar per second at 4°C. Direct measurements put a lower limit of k ≥ 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.

Chiral amines can be made by insertion of a carbene into an N–H bond using two-catalyst systems that combine a transition metal-based carbene-transfer catalyst and a chiral proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N–H insertion, but asymmetric protonation in an active site replete with proton sources is challenging. Here we describe engineered cytochrome P450 enzymes that catalyse carbene N–H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to 32,100 total turnovers, >99% yield and 98% e.e.). These enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this transformation and provide stereocontrol.A haem protein that serves as a dual-function catalyst capable of inserting a carbene into a N–H bond to form α-amino lactones has been reported. The enzyme catalyses both carbene transfer and the subsequent proton transfer in a single active site. This transformation can proceed at the gram scale with high efficiency and enantioselective control.

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s. Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.

Transition metal—catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C—H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C—C bond—forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450 BM3 that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature.

Cannabidiol (CBD) has been reported in medical applications for various indications. The enzymatic metabolism of CBD is not fully understood in the different routes of administration. This research aimed to identify the CBD metabolites after incubation of CBD with derived hepatocyte cells (HepG2), bronchial epithelial cells (NCI-H358), alveolar cells (A549), and alveolar macrophage cells (NR8383). A liquid chromatography–mass spectrometry technique was developed to quantify the CBD and its metabolites. Molecular docking was employed to evaluate the binding affinity of CBD with different cytochrome P-450 (CYP-450) enzymes and further predict the implication of drug–drug interactions. CBD and major metabolites of CBD were also docked with cannabinoid receptors. The results revealed that only HepG2 cells metabolized CBD to 7-hydroxy-CBD (7-OH-CBD) and 7-carboxy-CBD (7-COOH-CBD), whereas other respiratory cell lines and alveolar macrophages were found to have mainly CBD in the incubated samples without any metabolites. The CYP2C19 and CYP3A4 enzymes were responsible for CBD conversion to hydroxylated CBD metabolites. The 7-OH-CBD and 7-COOH-CBD metabolites were found to bind to cannabinoid receptors with different affinities. The relative abundance of CBD and major metabolites may indicate the potential route of CBD administration.

Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

Knowing how protein sequence maps to function (the “fitness landscape”) is critical for understanding protein evolution as well as for engineering proteins with new and useful properties. We demonstrate that the protein fitness landscape can be inferred from experimental data, using Gaussian processes, a Bayesian learning technique. Gaussian process landscapes can model various protein sequence properties, including functional status, thermostability, enzyme activity, and ligand binding affinity. Trained on experimental data, these models achieve unrivaled quantitative accuracy. Furthermore, the explicit representation of model uncertainty allows for efficient searches through the vast space of possible sequences. We develop and test two protein sequence design algorithms motivated by Bayesian decision theory. The first one identifies small sets of sequences that are informative about the landscape; the second one identifies optimized sequences by iteratively improving the Gaussian process model in regions of the landscape that are predicted to be optimized. We demonstrate the ability of Gaussian processes to guide the search through protein sequence space by designing, constructing, and testing chimeric cytochrome P450s. These algorithms allowed us to engineer active P450 enzymes that are more thermostable than any previously made by chimeragenesis, rational design, or directed evolution.

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.