Cytochrome P450 BM3 Variant Research Articles

Engineering of (−)‐Limonene‐3‐Hydroxylase to Enhance Its Regioselectivity for Biosynthesis of (−)‐Menthol

AbstractThere is high market demand for (−)‐menthol because of its unique aroma and cooling properties. The first bottleneck encountered in the biosynthesis of (−)‐menthol in Escherichia coli is the low hydroxylation efficiency of (−)‐limonene‐3‐hydroxylase. Few known enzymes can hydroxylate the third carbon atom of (−)‐limonene. In this work, initially, we searched for new enzymes and screened a library of cytochrome P450BM3 variants aiming to improve catalysis of this reaction, but did not identify any variants with improved activity or regioselectivity. Then, through protein engineering of a cytochrome P450 monooxygenase variant from Pseudomonas putida (PpcamY96F/V247L), we obtained variant PpcamF87W/Y96F/L244I/V247L/V396A with regioselectivity of 93% (starting from 70%) toward (−)‐limonene without losing activity. Because of the improvement in regioselectivity, its turnover number was 1.5 times that of the starting enzyme. We analyzed the reasons for the improvement through molecular dynamics simulations. The new variant also exhibited 95% regioselectivity toward (+)‐limonene. This enzyme variant can be applied in biosynthesis of (−)‐menthol.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants.

Cytochrome P450 BM3 monooxygenase is the topic of extensive research as many researchers have evolved this enzyme to generate a variety of products. However, the abundance of information on increasingly diversified variants of P450 BM3 that catalyze a broad array of chemistry is not in a format that enables easy extraction and interpretation. We present a database that categorizes variants by their catalyzed reactions and includes details about substrates to provide reaction context. This database of >1500 P450 BM3 variants is downloadable and machine-readable and includes instructions to maximize ease of gathering information. The database allows rapid identification of commonly reported substitutions, aiding researchers who are unfamiliar with the enzyme in identifying starting points for enzyme engineering. For those actively engaged in engineering P450 BM3, the database, along with this review, provides a powerful and user-friendly platform to understand, predict, and identify the attributes of P450 BM3 variants, encouraging the further engineering of this enzyme.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2 + 1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2 + 1] cyclopropanation, largely relies on the use of stereodefined olefins, which can require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here, we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450BM3 variant P411-INC-5185 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in the model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. P411-INC-5185 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to α-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.

The molecular mechanism of P450-catalyzed amination of the pyrrolidine derivative of lidocaine: insights from multiscale simulations.

Nitrogen heterocycles are key and prevalent motifs in drugs. Evolved variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium employ high-valent oxo-iron(iv) species to catalyze the synthesis of imidazolidine-4-ones via an intramolecular C–H amination. Herein, we use multi-scale simulations, including classical molecular dynamics (MD) simulations, quantum mechanical/molecular mechanical (QM/MM) calculations and QM calculations, to reveal the molecular mechanism of the intramolecular C–H amination of the pyrrolidine derivative of lidocaine bearing cyclic amino moieties catalyzed by the variant RP/FV/EV of P450BM3, which bears five mutations compared to wild type. Our calculations show that overall catalysis includes both the enzymatic transformation in P450 and non-enzymatic transformation in water solution. The enzymatic transformation involves the exclusive hydroxylation of the C–H bond of the pyrrolidine derivative of lidocaine, leading to the hydroxylated intermediate, during which the substrate radical would be bypassed. The following dehydration and C–N coupling reactions are found to be much favored in aqueous situation compared to that in the non-polar protein environment. The present findings expand our understanding of the P450-catalyzed C(sp3)–H amination reaction.

Altering the Regioselectivity of Cytochrome P450 BM3 Variant M13 toward Genistein through Protein Engineering and Variation of Reaction Conditions

The biocatalysts responsible for the enzymatic synthesis of hydroxygenisteins, derivatives of genistein with multiple activities, usually show regioselective promiscuity, hydroxylating genistein to form a mixture of multiple products, which, in turn, results in a cumbersome separation and purification. Hence, it is highly desired to explore the underlying mechanism regulating the regioselectivity of hydroxylases. M13 is a variant of cytochrome P450 BM3 with oxidant activity toward genistein. Herein, genistein was demonstrated to be hydroxylated by M13 to form a mixture of 3′-hydroxygenistein (3′-OHG) and 8-hydroxygenistein (8-OHG), each giving 4% conversion with a ratio of 1:1. Protein engineering toward M13 was thus performed to improve its regioselectivity. When isoleucine at position 86 was mutated into cysteine, the resultant variant M13I86C displayed improved regioselectivity toward 3′-OHG with an increased conversion of 8.5%. The double mutation M13I86CP18W further boosted the conversion of 3′-OHG to 9.6%, and the ratio of 3′-OHG to 8-OHG increased to 12:1. Conversely, both CoCl2 and glucose 6-phosphate (G6P) could lead to more 8-OHG. When Co2+ reached 37.5 mM, M13I86CP18W could give an 8-OHG conversion of 22.4%. The maximal ratio of 8-OHG to 3′-OHG reached 130 when 62.5 mM Co2+ was included in the reaction mixture. With the increase of G6P from 10 to 40 mM, the conversion of M13I86CP18W to 8-OHG gradually increased to 22.6%, while the conversion to 3′-OHG decreased to 6%. Thus, both intrinsic residues and external reaction conditions can affect the regiospecificity of M13, which laid the foundation for the selection of suitable biocatalysts for the hydroxylation of genistein.

Peroxide-Driven Hydroxylation of Small Alkanes Catalyzed by an Artificial P450BM3 Peroxygenase System

We report the selective hydroxylation of small alkanes with H2O2 catalyzed by an artificial P450 peroxygenase system generated from engineered cytochrome P450BM3 variants in assistance with dual-fu...

Hydroxylation of Eleuthoside Synthetic Intermediates by P450BM3 (CYP102A1)

A seven‐step synthesis of the first tricyclic intermediate in Danishefsky's total synthesis of eleutherobin is reported with Wittig homologation and sequential additions of metallated furan derivatives to aldehydes as key steps. Aiming to prepare hydroxylated eleutherobin analogues for cytotoxicity screening, the furan diol epimers 6 and precursors 1 and 2 were exposed to a 48‐member panel of engineered cytochrome P450BM3 variants. Both furan‐containing substrates reacted solely at the furan ring, one resulting in overall biocatalytic Achmatowicz reaction. Selective hydroxylation at four separate sp3 C–H centres was achieved in the substrates lacking the furan component. P450BM3‐catalysed hydroxylation of racemic substrates can proceed with kinetic resolution; in the case reported here, the 2°‐allylic alcohol product 10 was generated with an 82:18 enantiomeric ratio.

Cross-linked cytochrome P450 BM3 aggregates promoted by Ru(II)-diimine complexes bearing aldehyde groups

Cross-linked enzyme aggregate (CLEA) methodology has been applied to immobilize cytochrome P450 BM3 variants (F87A and 21B3) with peroxygenase activity. Several Ru(II)-diimine complexes were found to be suitable cross-linking agents, surpassing the traditional glutaraldehyde and dextran aldehyde. They offer modular numbers of aldehyde functionalities and a more rigid framework than their organic counterparts. The F87A CLEAs display significant activity loss compared to the protein in solution. Meanwhile, for the 21B3 CLEAs, high activity recovery (up to 95%) is obtained. In order to minimize enzyme leaching from the CLEA, sodium cyanoborohydride was used to reduce the CLEAs imine bonds. The reduced CLEAs were active for several rounds of reactions leading to an overall increase in protein activity of 170% compared to the free protein in solution.

A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants

The 5′-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5′-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5′-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5′-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5′-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450BM3 that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatom-substituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.

Exploring PTDH-P450BM3 Variants for the Synthesis of Drug Metabolites.

The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.

Frontispiece: Mechanistic Study of the Stereoselective Hydroxylation of [2‐2H1,3‐2H1]Butanes Catalyzed by Cytochrome P450 BM3 Variants

Frontispiece: Mechanistic Study of the Stereoselective Hydroxylation of [2‐²H₁,3‐²H₁]Butanes Catalyzed by Cytochrome P450 BM3 Variants

Mechanistic Study of the Stereoselective Hydroxylation of [2-2 H1 ,3-2 H1 ]Butanes Catalyzed by Cytochrome P450 BM3 Variants.

Engineered bacterial cytochrome P450s are noted for their ability in the oxidation of inert small alkanes. Cytochrome P450 BM3 L188P A328F (BM3 PF) and A74E L188P A328F (BM3 EPF) variants are able to efficiently oxidize n-butane to 2-butanol. Esterification of the 2-butanol derived from this reaction mediated by the aforementioned two mutants gives diastereomeric excesses (de) of -56±1 and -52±1 %, respectively, with the preference for the oxidation occurring at the C-HS bond. When tailored (2R,3R)- and (2S,3S)-[2-2 H1 ,3-2 H1 ]butane probes are employed as substrates for both variants, the obtained de values from (2R,3R)-[2-2 H1 ,3-2 H1 ]butane are -93 and -92 % for BM3 PF and EPF, respectively; whereas the obtained de values from (2S,3S)-[2-2 H1 ,3-2 H1 ]butane are 52 and 56 % in the BM3 PF and EPF systems, respectively. The kinetic isotope effects (KIEs) for the oxidation of (2R,3R)-[2-2 H1 ,3-2 H1 ]butane are 7.3 and 7.8 in BM3 PF and EPF, respectively; whereas KIEs for (2S,3S)-[2-2 H1 ,3-2 H1 ]butanes are 18 and 25 in BM3 PF and EPF, respectively. The discrepancy in KIEs obtained from the two substrates supports the two-state reactivity (TSR) that is proposed for alkane oxidation in cytochrome P450 systems. Moreover, for the first time, experimental evidence for tunneling in the oxidation mediated by P450 is given through the oxidation of the C-HR bond in (2S,3S)-[2-2 H1 ,3-2 H1 ]butane.

Synthesis of Imidazolidin-4-ones via a Cytochrome P450-Catalyzed Intramolecular C–H Amination

Expanding Nature’s catalytic repertoire to include reactions important in synthetic chemistry opens new opportunities for biocatalysis. An intramolecular C–H amination route to imidazolidin-4-ones via α-functionalization of 2-aminoacetamides catalyzed by evolved variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium has been developed. Screening of a library of ca. 100 variants based on four template mutants with enhanced activity for the oxidation of unnatural substrates and preparative scale reactions in vitro and in vivo show that the enzymes give up to 98% isolated yield of cyclization products for diverse substrates. 2-Aminoacetamides with one- and two-ring cyclic amines bearing substituents and aliphatic, alicyclic, and substituted aromatic amides are cyclized. Regiodivergent C–H amination was achieved at benzylic and nonbenzylic positions in a tetrahydroisoquinolinyl substrate by the use of different mutants. This C–H amination reaction offers a scalable route to imidazolidin-4-ones wit...

Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant.

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.

Use of a genetically encoded hydrogen peroxide sensor for whole cell screening of enzyme activity

We report the use of HyPer, a genetically encoded, fluorescent sensor that reacts with hydrogen peroxide (H2O2), in a novel screen to engineer enzymes for enhanced production of H2O2. We co-expressed HyPer with cytochrome P450 BM3 variants and, using HyPer's ratiometric signal, found variants that produce greater amounts of H2O2 than the wild-type enzyme through the leakage reaction. The screen avoids lysis procedures and the addition of reagents to assay intracellular contents. Less laborious screening procedures will be useful in engineering more powerful H2O2 generators as tools in quantitative redox biology, and increasing the utility of enzymes that produce H2O2 as a by-product alongside a valuable compound.

Enzyme-Controlled Regiodivergent C–H Amination

Significance: Arnold and co-workers report an enzyme-catalyzed asymmetric and regiodivergent C–H amination. By modifying the active site of P411BM3, a cytochrome P450BM3 variant in which the axial position of the iron-heme prosthetic group is ligated by serine instead of cysteine, two variants enabling different regioselectivity were obtained. One allowed the selective amination of the energetically more favored benzylic position, while the other was selective for the homobenzylic position of 2,5-disubstituted benzenesulfonyl azides. Comment: Due to the availability of a weaker benzylic C–H bond, direct C–H amination of homobenzylic positions are a challenge in small-molecule catalysis. Breaking the C–H bond is kinetically controlled, and therefore, this can in some cases be overcome by variation of the steric properties of the used ligands. However, genetically engineered enzymes are an attractive alternative for regiodivergent C–H amination, enabling access to the desired products in good to excellent regioand enantioselectivities. R1

Enzyme-controlled nitrogen-atom transfer enables regiodivergent C-H amination.

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.

Non-natural olefin cyclopropanation catalyzed by diverse cytochrome P450s and other hemoproteins.

Recent work has shown that engineered variants of cytochrome P450BM3 (CYP102A1) efficiently catalyze non-natural reactions, including carbene and nitrene transfer reactions. Given the broad substrate range of natural P450 enzymes, we set out to explore if this diversity could be leveraged to generate a broad panel of new catalysts for olefin cyclopropanation (i.e., carbene transfer). Here, we took a step towards this goal by characterizing the carbene transfer activities of four new wild-type P450s that have different native substrates. All four were active and exhibited a range of product selectivities in the model reaction: cyclopropanation of styrene by using ethyl diazoacetate (EDA). Previous work on P450BM3 demonstrated that mutation of the axial coordinating cysteine, universally conserved among P450 enzymes, to a serine residue, increased activity for this non-natural reaction. The equivalent mutation in the selected P450s was found to activate carbene transfer chemistry both in vitro and in vivo. Furthermore, serum albumins complexed with hemin were also found to be efficient in vitro cyclopropanation catalysts.

Enantioselective imidation of sulfides via enzyme-catalyzed intermolecular nitrogen-atom transfer.

Engineering enzymes with novel reaction modes promises to expand the applications of biocatalysis in chemical synthesis and will enhance our understanding of how enzymes acquire new functions. The insertion of nitrogen-containing functional groups into unactivated C–H bonds is not catalyzed by known enzymes but was recently demonstrated using engineered variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium. Here, we extend this novel P450-catalyzed reaction to include intermolecular insertion of nitrogen into thioethers to form sulfimides. An examination of the reactivity of different P450BM3 variants toward a range of substrates demonstrates that electronic properties of the substrates are important in this novel enzyme-catalyzed reaction. Moreover, amino acid substitutions have a large effect on the rate and stereoselectivity of sulfimidation, demonstrating that the protein plays a key role in determining reactivity and selectivity. These results provide a stepping stone for engineering more complex nitrogen-atom-transfer reactions in P450 enzymes and developing a more comprehensive biocatalytic repertoire.