Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-ß-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes.

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from "off-the-shelf" iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M-1 s-1, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with "off-the-shelf" reagents.

Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with 'rule-of-three' properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Enantio- and Diastereodivergent Cyclopropanation of Allenes by Directed Evolution of an Iridium-Containing Cytochrome.

Alkylidene cyclopropanes (ACPs) are valuable synthetic intermediates because of their constrained structure and opportunities for further diversification. Although routes to ACPs are known, preparations of ACPs with control of both the configuration of the cyclopropyl (R vs S) group and the geometry of the alkene (E vs Z) are unknown. We describe enzymatic cyclopropanation of allenes with ethyl diazoacetate (EDA) catalyzed by an iridium-containing cytochrome (Ir(Me)-CYP119) that controls both stereochemical elements. Two mutants of Ir(Me)-CYP119 identified by 6-codon (6c, VILAFG) saturation mutagenesis catalyze the formation of (E)-ACPs with -93% to >99% ee and >99:1 E/Z ratio with just three rounds of 96 mutants. By four additional rounds of mutagenesis, an enzyme variant was identified that forms (Z)-ACPs with up to 94% ee and a 28:72 E/Z ratio. Computational studies show that the orientation of the carbene unit dictated by the mutated positions accounts for the stereoselectivity.

Reversing the Enantioselectivity of Enzymatic Carbene N-H Insertion Through Mechanism-Guided Protein Engineering.

We report a computationally driven approach to access enantiodivergent enzymatic carbene N-H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N-H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er).

Protoglobin-Catalyzed Formation of cis-Trifluoromethyl-Substituted Cyclopropanes by Carbene Transfer.

Trifluoromethyl-substituted cyclopropanes (CF3 -CPAs) constitute an important class of compounds for drug discovery. While several methods have been developed for synthesis of trans-CF3 -CPAs, stereoselective production of corresponding cis-diastereomers remains a formidable challenge. We report a biocatalyst for diastereo- and enantio-selective synthesis of cis-CF3 -CPAs with activity on a variety of alkenes. We found that an engineered protoglobin from Aeropyrnum pernix (ApePgb) can catalyze this unusual reaction at preparative scale with low-to-excellent yield (6-55 %) and enantioselectivity (17-99 % ee), depending on the substrate. Computational studies revealed that the steric environment in the active site of the protoglobin forced iron-carbenoid and substrates to adopt a pro-cis near-attack conformation. This work demonstrates the capability of enzyme catalysts to tackle challenging chemistry problems and provides a powerful means to expand the structural diversity of CF3 -CPAs for drug discovery.

Selective Biocatalytic N-Methylation of Unsaturated Heterocycles.

Methods for regioselective N-methylation and -alkylation of unsaturated heterocycles with "off the shelf" reagents are highly sought-after. This reaction could drastically simplify synthesis of privileged bioactive molecules. Here we report engineered and natural methyltransferases for challenging N-(m)ethylation of heterocycles, including benzimidazoles, benzotriazoles, imidazoles and indazoles. The reactions are performed through a cyclic enzyme cascade that consists of two methyltransferases using only iodoalkanes or methyl tosylate as simple reagents. This method enables the selective synthesis of important molecules that are otherwise difficult to access, proceeds with high regioselectivity (r.r. up to >99 %), yield (up to 99 %), on a preparative scale, and with nearly equimolar concentrations of simple starting materials.

Enzymatic Control over Reactive Intermediates Enables Direct Oxidation of Alkenes to Carbonyls by a P450 Iron-Oxo Species.

The aerobic oxidation of alkenes to carbonyls is an important and challenging transformation in synthesis. Recently, a new P450-based enzyme (aMOx) has been evolved in the laboratory to directly oxidize styrenes to their corresponding aldehydes with high activity and selectivity. The enzyme utilizes a heme-based, high-valent iron-oxo species as a catalytic oxidant that normally epoxidizes alkenes, similar to other catalysts. How the evolved aMOx enzyme suppresses the commonly preferred epoxidation and catalyzes direct carbonyl formation is currently not well understood. Here, we combine computational modelling together with mechanistic experiments to study the reaction mechanism and unravel the molecular basis behind the selectivity achieved by aMOx. Our results describe that although both pathways are energetically accessible diverging from a common covalent radical intermediate, intrinsic dynamic effects determine the strong preference for epoxidation. We discovered that aMOx overrides these intrinsic preferences by controlling the accessible conformations of the covalent radical intermediate. This disfavors epoxidation and facilitates the formation of a carbocation intermediate that generates the aldehyde product through a fast 1,2-hydride migration. Electrostatic preorganization of the enzyme active site also contributes to the stabilization of the carbocation intermediate. Computations predicted that the hydride migration is stereoselective due to the enzymatic conformational control over the intermediate species. These predictions were corroborated by experiments using deuterated styrene substrates, which proved that the hydride migration is cis- and enantioselective. Our results demonstrate that directed evolution tailored a highly specific active site that imposes strong steric control over key fleeting biocatalytic intermediates, which is essential for accessing the carbonyl forming pathway and preventing competing epoxidation.

Changes in Protonation States of In-Pathway Residues can Alter Ligand Binding Pathways Obtained From Spontaneous Binding Molecular Dynamics Simulations.

Protein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 Å away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations.

Engineered P450 Atom-Transfer Radical Cyclases are Bifunctional Biocatalysts: Reaction Mechanism and Origin of Enantioselectivity.

New-to-nature radical biocatalysis has recently emerged as a powerful strategy to tame fleeting open-shell intermediates for stereoselective transformations. In 2021, we introduced a novel metalloredox biocatalysis strategy that leverages the innate redox properties of the heme cofactor of P450 enzymes, furnishing new-to-nature atom-transfer radical cyclases (ATRCases) with excellent activity and stereoselectivity. Herein, we report a combined computational and experimental study to shed light on the mechanism and origins of enantioselectivity for this system. Molecular dynamics and quantum mechanics/molecular mechanics (QM/MM) calculations revealed an unexpected role of the key beneficial mutation I263Q. The glutamine residue serves as an essential hydrogen bond donor that engages with the carbonyl moiety of the substrate to promote bromine atom abstraction and enhance the enantioselectivity of radical cyclization. Therefore, the evolved ATRCase is a bifunctional biocatalyst, wherein the heme cofactor enables atom-transfer radical biocatalysis, while the hydrogen bond donor residue further enhances the activity and enantioselectivity. Unlike many enzymatic stereocontrol rationales based on a rigid substrate binding model, our computations demonstrate a high degree of rotational flexibility of the allyl moiety in an enzyme-substrate complex and succeeding intermediates. Therefore, the enantioselectivity is controlled by the radical cyclization transition states rather than the substrate orientation in ground-state complexes in the preceding steps. During radical cyclization, anchoring effects of the Q263 residue and steric interactions with the heme cofactor concurrently control the π-facial selectivity, allowing for highly enantioselective C-C bond formation. Our computational findings are corroborated by experiments with ATRCase mutants generated from site-directed mutagenesis.

Directed evolution of nonheme iron enzymes to access abiological radical-relay C(sp3)-H azidation.

We report the reprogramming of nonheme iron enzymes to catalyze an abiological C(sp3)‒H azidation reaction through iron-catalyzed radical relay. This biocatalytic transformation uses amidyl radicals as hydrogen atom abstractors and Fe(III)‒N3 intermediates as radical trapping agents. We established a high-throughput screening platform based on click chemistry for rapid evolution of the catalytic performance of identified enzymes. The final optimized variants deliver a range of azidation products with up to 10,600 total turnovers and 93% enantiomeric excess. Given the prevalence of radical relay reactions in organic synthesis and the diversity of nonheme iron enzymes, we envision that this discovery will stimulate future development of metalloenzyme catalysts for synthetically useful transformations unexplored by natural evolution.

An Enzymatic Platform for Primary Amination of 1-Aryl-2-alkyl Alkynes.

Propargyl amines are versatile synthetic intermediates with numerous applications in the pharmaceutical industry. An attractive strategy for efficient preparation of these compounds is nitrene propargylic C(sp3)-H insertion. However, achieving this reaction with good chemo-, regio-, and enantioselective control has proven to be challenging. Here, we report an enzymatic platform for the enantioselective propargylic amination of alkynes using a hydroxylamine derivative as the nitrene precursor. Cytochrome P450 variant PA-G8 catalyzing this transformation was identified after eight rounds of directed evolution. A variety of 1-aryl-2-alkyl alkynes are accepted by PA-G8, including those bearing heteroaromatic rings. This biocatalytic process is efficient and selective (up to 2610 total turnover number (TTN) and 96% ee) and can be performed on preparative scale.

Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase.

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved k cat/K m values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.

Dual-function enzyme catalysis for enantioselective carbon-nitrogen bond formation.

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 shared mechanistic pathway for pyridoxal phosphate-dependent arginine oxidases.

The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.

The Unexplored Importance of Fleeting Chiral Intermediates in Enzyme-Catalyzed Reactions.

Decades of extensive research efforts by biochemists, organic chemists, and protein engineers have led to an understanding of the basic mechanisms of essentially all known types of enzymes, but in a formidable number of cases an essential aspect has been overlooked. The occurrence of short-lived chiral intermediates formed by symmetry-breaking of prochiral precursors in enzyme catalyzed reactions has been systematically neglected. We designate these elusive species as fleeting chiral intermediates and analyze such crucial questions as "Do such intermediates occur in homochiral form?" If so, what is the absolute configuration, and why did Nature choose that particular stereoisomeric form, even when the isolable final product may be achiral? Does the absolute configuration of a chiral product depend in any way on the absolute configuration of the fleeting chiral precursor? How does this affect the catalytic proficiency of the enzyme? If these issues continue to be unexplored, then an understanding of the mechanisms of many enzyme types remains incomplete. We have systematized the occurrence of these chiral intermediates according to their structures and enzyme types. This is followed by critical analyses of selected case studies and by final conclusions and perspectives. We hope that the fascinating concept of fleeting chiral intermediates will attract the attention of scientists, thereby opening an exciting new research field.

Origin and Control of Chemoselectivity in Cytochrome c Catalyzed Carbene Transfer into Si-H and N-H bonds.

A cytochrome c heme protein was recently engineered to catalyze the formation of carbon-silicon bonds via carbene insertion into Si-H bonds, a reaction that was not previously known to be catalyzed by a protein. High chemoselectivity toward C-Si bond formation over competing C-N bond formation was achieved, although this trait was not screened for during directed evolution. Using computational and experimental tools, we now establish that activity and chemoselectivity are modulated by conformational dynamics of a protein loop that covers the substrate access to the iron-carbene active species. Mutagenesis of residues computationally predicted to control the loop conformation altered the protein's chemoselectivity from preferred silylation to preferred amination of a substrate containing both N-H and Si-H functionalities. We demonstrate that information on protein structure and conformational dynamics, combined with knowledge of mechanism, leads to understanding of how non-natural and selective chemical transformations can be introduced into the biological world.

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics.

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 ß-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.

Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics.

Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent useful tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244, and/or Leu295, that alter the native selectivity, step sequence, and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity, enabling catalyst-controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.

Machine Learning Enables Selection of Epistatic Enzyme Mutants for Stability Against Unfolding and Detrimental Aggregation.

Machine learning (ML) has pervaded most areas of protein engineering, including stability and stereoselectivity. Using limonene epoxide hydrolase as the model enzyme and innov'SAR as the ML platform, comprising a digital signal process, we achieved high protein robustness that can resist unfolding with concomitant detrimental aggregation. Fourier transform (FT) allows us to take into account the order of the protein sequence and the nonlinear interactions between positions, and thus to grasp epistatic phenomena. The innov'SAR approach is interpolative, extrapolative and makes outside-the-box, predictions not found in other state-of-the-art ML or deep learning approaches. Equally significant is the finding that our approach to ML in the present context, flanked by advanced molecular dynamics simulations, uncovers the connection between epistatic mutational interactions and protein robustness.