Engineering Cytochrome P450 BM3 for Terminal Alkane Hydroxylation

Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium‐chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active‐site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77‐9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane‐specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation.     

Specific Modulation of Protein Activity by Using a Bioorthogonal Reaction

Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a “click” reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z‐containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active‐site occlusion and allosteric mechanisms. We have termed this a “clickable magic bullet” strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.     

Towards Understanding Directed Evolution: More than Half of All Amino Acid Positions Contribute to Ionic Liquid Resistance of Bacillus subtilis Lipase A

Ionic liquids (ILs) are attractive (co‐)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]‐based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50–69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6–13 % of substitutions led to improved resistance. Among these, 66–95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.     

Semirational Protein Engineering of CYP153AM.aq.‐CPRBM3 for Efficient Terminal Hydroxylation of Short‐ to Long‐Chain Fatty Acids

The regioselective terminal hydroxylation of alkanes and fatty acids is of great interest in a variety of industrial applications, such as in cosmetics, in fine chemicals, and in the fragrance industry. The chemically challenging activation and oxidation of non‐activated C?H bonds can be achieved with cytochrome P450 enzymes. CYP153A_M.aq.‐CPR_BM3 is an artificial fusion construct consisting of the heme domain from Marinobacter aquaeolei and the reductase domain of CYP102A1 from Bacillus megaterium. It has the ability to hydroxylate medium‐ and long‐chain fatty acids selectively at their terminal positions. However, the activity of this interesting P450 construct needs to be improved for applications in industrial processes. For this purpose, the design of mutant libraries including two consecutive steps of mutagenesis is demonstrated. Targeted positions and residues chosen for substitution were based on semi‐rational protein design after creation of a homology model of the heme domain of CYP153A_M.aq., sequence alignments, and docking studies. Site‐directed mutagenesis was the preferred method employed to address positions within the binding pocket, whereas diversity was created with the aid of a degenerate codon for amino acids located at the substrate entrance channel. Combining the successful variants led to the identification of a double variant—G307A/S233G—that showed alterations of one position within the binding pocket and one position located in the substrate access channel. This double variant showed twofold increased activity relative to the wild type for the terminal hydroxylation of medium‐chain‐length fatty acids. This variant furthermore showed improved activity towards short‐ and long‐chain fatty acids and enhanced stability in the presence of higher concentrations of fatty acids.     

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

We recently developed an artificial P450–H₂O₂ system assisted by dual-functional small molecules (DFSMs) to modify the P450BM3 monooxygenase into its peroxygenase mode, which could be widely used for the oxidation of non-native substrates. Aiming to further improve the DFSM-facilitated P450–H₂O₂ system, a series of novel DFSMs having various unnatural amino acid groups was designed and synthesized, based on the co-crystal structure of P450BM3 and a typical DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine, in this study. The size and hydrophobicity of the amino acid residue in the DFSM drastically affected the catalytic activity (up to 5-fold), stereoselectivity, and regioselectivity of the epoxidation and hydroxylation reactions. Docking simulations illustrated that the differential catalytic ability among the DFSMs is closely related to the binding affinity and the distance between the catalytic group and heme iron. This study not only enriches the DFSM toolbox to provide more options for utilizing the peroxide-shunt pathway of cytochrome P450BM3, but also sheds light on the great potential of the DFSM-driven P450 peroxygenase system in catalytic applications based on DFSM tunability.     

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single‐ versus Double‐Code Saturation Mutagenesis

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single‐code saturation mutagenesis (SCSM) and triple‐code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double‐code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)‐ or (S,S)‐cyclohexane‐1,2‐diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R‐ and S,S‐selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.     

Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site‐Directed Mutagenesis

Previous studies on cytochrome P450 monooxygenases (CYP) from family 154 reported their substrate promiscuity and high activity. Hence, herein, the uncharacterized family member CYP154F1 is described. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8–10 atoms. In contrast to thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and lower activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in the active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids led to up to tenfold higher conversion rates of small substrates, such as geraniol. Replacement of T234 by small hydrophobic amino acids, valine or alanine, resulted in mutants with extended substrate spectra. These mutants are able to convert some of the larger substrates of CYP154E1, such as (E)‐stilbene and (+)‐nootkatone.     

Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis

The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450_BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450_BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450_BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (T_opt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450_BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.     

Structural Insights into the Recovery of Aldolase Activity in N‐Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ‐Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK_a of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.     

Catalytic Asymmetric Synthesis of Functionalized α,α‐Disubstituted α‐Amino Acid Derivatives from Racemic Unprotected α‐Amino Acids via in‐situ Generated Azlactones

Masked and activated highly enantioenriched α,α‐disubstituted α‐amino acids with an additional adjacent stereocenter were formed by a tandem reaction involving five steps using racemic unprotected amino acid substrates. Key step is the 1,4‐addition of in‐situ generated azlactones to a broad number of enones. The products of this step‐economic route can, e.g., be useful for a divergent and rapid access to biologically interesting unnatural glutamic acid derivatives.     

P450 BM3‐Catalyzed Regio‐ and Stereoselective Hydroxylation Aiming at the Synthesis of Phthalides and Isocoumarins

Cytochrome P450 BM3 monooxygenases are able to catalyze the regio‐ and stereoselective oxygenation of a broad range of substrates, with promising potential for synthetic applications. To study the suitability of P450 BM3 variants for stereoselective benzylic hydroxylation of 2‐alkylated benzoic acid esters, the biotransformation of methyl 2‐ethylbenzoate, resulting in both enantiomeric forms of 3‐methylphthalide, was investigated. In the case of methyl 2‐propylbenzoate as a substrate the regioselectivity of the reaction was shifted towards β‐hydroxylation, resulting in the synthesis of enantioenriched R‐ and S‐configured 3‐methylisochroman‐1‐one. The potential of P450 BM3 variants for regio‐ and stereoselective synthesis of phthalides and isocoumarins offers a new route to a class of compounds that are valuable synthons for a variety of natural compounds.     

Natural Diversity to Guide Focused Directed Evolution

Simultaneous multiple site‐saturation mutagenesis was performed at four active‐site positions of an esterase from Pseudomonas fluorescens to improve its ability to convert 3‐phenylbutyric acid esters (3‐PBA) in an enantioselective manner. Based on an appropriate codon choice derived from a structural alignment of 1751 sequences of α/β‐hydrolase fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivalent positions. Thus, the number of mutants to be screened could be substantially reduced while the number of functionally intact variants was increased. Whereas the wild‐type esterase showed only marginal activity and poor enantioselectivity (E_true=3.2) towards 3‐PBA‐ethyl ester, a significant number of hits with improved rates (up to 240‐fold) and enantioselectivities (up to E_true=80) were identified in these “smart” libraries.     

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

The Escherichia coli esterase YbfF displays high activity towards 1,2‐O‐isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R‐enantiomer of these substrates, producing the S‐enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R‐enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2‐fold higher enantioselectivity than wild‐type YbfF, with an E=38 for IPG butyrate and an E=77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.     

Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.     

Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone

CYP106A2 is known as a 15β‐hydroxylase, but also shows minor 11α‐hydroxylase activity for progesterone. 11α‐Hydroxyprogesterone is an important pharmaceutical compound with anti‐androgenic and blood‐pressure‐regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site‐directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site‐directed mutagenesis of active‐site residues (Lisurek et al. ChemBioChem 2008 , 9, 1439–1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α‐hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site‐directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α‐hydroxylase selectivities and activities relative to the WT (14.3‐, 12.6‐, and 11.8‐fold increases in selectivity and 39.3‐, 108‐, and 24.4‐ in k_cat/K_m). In the last step of the study, the best mutants were applied in a whole‐cell biotransformation. In these experiments the production (percentage) of 15β‐hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α‐hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.     

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T₅₀ >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.     

Regioselectivity and Activity of Cytochrome P450 BM‐3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide

Cytochrome P450 BM‐3 (EC 1.14.14.1) is a monooxygenase that utilizes NADPH and dioxygen to hydroxylate fatty acids at subterminal positions. The enzyme is also capable of functioning as a peroxygenase in the same reaction, by utilizing hydrogen peroxide in place of the reductase domain, cofactor and oxygen. As a starting point for developing a practically useful hydroxylation biocatalyst, we compare the activity and regioselectivity of wild‐type P450 BM‐3 and its F87A mutant on various fatty acids. Neither enzyme catalyzes terminal hydroxylation under any of the conditions studied. While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position. The H₂O₂‐driven reactions with either the full‐length BM‐3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O₂.     

Increasing the Reaction Rate of Hydroxynitrile Lyase from Hevea brasiliensis toward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single‐amino‐acid‐substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL‐L121Y, with a k_cat 4.2 times higher and high enantioselectivity. Site‐saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.     

Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes

Three semi-rational approaches, combinatorial site-saturation mutagenesis (CSSM) using a reduced amino acid set and two libraries based on C(orbit) and CRAM computational design algorithms targeting up to 10 active site residues, were used to engineer cytochrome P450 BM3 to demethylate dimethyl ether and hydroxylate propane and ethane. These small libraries (343-1028 variants) were all enriched with respect to the fraction functional and maximal activities compared with a random mutagenesis library and individual site-saturation libraries targeting the same residues. Despite high average amino acid substitution levels of 2.6, 5 and 7.5, the CSSM, C(orbit) and CRAM libraries had at least 75% of library members properly folded. Propane- and ethane-hydroxylating P450 BM3 variants were identified using all three mutagenesis approaches, with as few as two amino acid substitutions. The library designed using the CRAM algorithm, which sought to reduce the size of the binding pocket, produced both a higher number of active variants and variants supporting the greatest number of catalytic turnovers. The most active variant E32 supports 16 800 propane turnovers at 36% coupling, which rivals the activity of variants obtained after 10-12 rounds of directed evolution using random and site-saturation mutagenesis. None of the variants in this study achieved the complete re-specialization for propane hydroxylation (including 93% coupling) previously obtained via multiple rounds of mutagenesis and screening. However, these semi-rational approaches allowed for large jumps in sequence space to variants with the desired functions.     

Novel Furin Inhibitors with Potent Anti‐infectious Activity

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4‐guanidinomethyl‐phenylacteyl‐Arg‐Tle‐Arg‐4‐amidinobenzylamide (MI‐1148) inhibits furin with a K_i value of 5.5 pM . The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI‐1148 was crystallized in complex with human furin. Its N‐terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short‐term treatment of acute infectious diseases.