Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C? H bonds mainly in a stereo‐ and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3‐keto‐4‐ene steroids mainly at position 15. We expressed this enzyme together with the electron‐transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme‐coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450‐catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2‐catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18‐fold to yield an absolute productivity up to 5.5 g L ^?1 d^?1. Product extraction with absorber resins allowed the recovery of quantitative amounts of 15β‐OH‐progesterone and 15β‐OH‐testosterone and also the reuse of the biocatalyst.     

Protein Engineering of the Progesterone Hydroxylating P450‐Monooxygenase CYP17A1 Alters Its Regioselectivity

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17?C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active‐site residues were identified and systematically mutated to alanine. In whole‐cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α‐hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.     

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.     

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.     

Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C₁₈ polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.     

Tracking Down a New Steroid‐Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233‐SM

CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11‐ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α‐hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16‐hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α‐dihydroxyprogesterone and 6β,16α‐dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.     

Hijacking Chemical Reactions of P450 Enzymes for Altered Chemical Reactions and Asymmetric Synthesis

Cytochrome P450s are heme-containing enzymes capable of the oxidative transformation of a wide range of organic substrates. A protein scaffold that coordinates the heme iron, and the catalytic pocket residues, together, determine the reaction selectivity and regio- and stereo-selectivity of the P450 enzymes. Different substrates also affect the properties of P450s by binding to its catalytic pocket. Modulating the redox potential of the heme by substituting iron-coordinating residues changes the chemical reaction, the type of cofactor requirement, and the stereoselectivity of P450s. Around hundreds of P450s are experimentally characterized, therefore, a mechanistic understanding of the factors affecting their catalysis is increasingly vital in the age of synthetic biology and biotechnology. Engineering P450s can enable them to catalyze a variety of chemical reactions viz. oxygenation, peroxygenation, cyclopropanation, epoxidation, nitration, etc., to synthesize high-value chiral organic molecules with exceptionally high stereo- and regioselectivity and catalytic efficiency. This review will focus on recent studies of the mechanistic understandings of the modulation of heme redox potential in the engineered P450 variants, and the effect of small decoy molecules, dual function small molecules, and substrate mimetics on the type of chemical reaction and the catalytic cycle of the P450 enzymes.     

Altering the regioselectivity of cytochrome P450 CYP102A3 of Bacillus subtilis by using a new versatile assay system

A novel monooxygenase (CYP102A3) has been discovered within the Bacillus subtilis genome that reveals a similarity of 76 % to the well-known cytochrome P450 BM-3 of B. megaterium (CYP102A1). Both enzymes are natural fusion proteins consisting of a heme domain and a FAD/FMN-reductase domain. Because of their high turnover rates, these biocatalysts are of special interest for industrial applications, but show only limited regioselectivity. In this work, the regioselectivity of CYP102A3 was changed by directed evolution and protein design to hydroxylate substrates not only in different subterminal, but also to a high extent, in terminal carbon chain positions. To enable a high-throughput screening procedure, a very versatile assay was developed that is capable of discriminating between terminal and subterminal hydroxylation of carbon chains. A double mutant of CYP102A3 was obtained that produces 48 % octan-1-ol as the main product of the reaction.     

Altering the Regioselectivity of a Nitroreductase in the Synthesis of Arylhydroxylamines by Structure‐Based Engineering

Nitroreductases have great potential for the highly efficient reduction of aryl nitro compounds to arylhydroxylamines. However, regioselective reduction of the desired nitro group in polynitroarenes is still a challenge. Here, we describe the structure‐based engineering of Escherichia coli nitroreductase NfsB to alter its regioselectivity, in order to achieve reduction of a target nitro group. When 2,4‐dinitrotoluene was used as the substrate, the wild‐type enzyme regioselectively reduced the 4‐NO₂ group, but the T41L/N71S/F124W mutant primarily reduced the 2‐NO₂ group, without loss of activity. The crystal structure of T41L/N71S/F124W and docking experiments indicated that the regioselectivity change (from 4‐NO₂ to 2‐NO₂) might result from the increased hydrophobicity of residues 41 and 124 (proximal to FMN) and conformational changes in residues 70 and 124.     

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti‐tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self‐sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild‐type enzyme converts (4R)‐limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio‐, stereo‐ and chemoselectivity, compared to wild‐type. From this library the variant with the highest perillyl alcohol ratio was fine‐tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)‐limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity.     

Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

We present a novel fragment‐based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate‐like ligands for a protein class of interest. Second, the library can be screened for binding to a novel putative ligand‐binding protein from the same or similar class, and the characterization of hits provides insight into the basis of ligand recognition, selectivity, and function at the substrate level. As a proof‐of‐concept, we applied the biofragments approach to the functionally uncharacterized Mycobacterium tuberculosis (Mtb) cytochrome P450 isoform, CYP126. This led to the development of a tailored CYP biofragment library with notable 3D characteristics and a significantly higher screening hit rate (14 %) than standard drug‐like fragment libraries screened previously against Mtb CYP121 and 125 (4 % and 1 %, respectively). Biofragment hits were identified that make both substrate‐like type‐I and inhibitor‐like type‐II interactions with CYP126. A chemical‐fingerprint‐based substrate model was built from the hits and used to search a virtual TB metabolome, which led to the discovery that CYP126 has a strong preference for the recognition of aromatics and substrate‐like type‐I binding of chlorophenol moieties within the active site near the heme. Future catalytic analyses will be focused on assessing CYP126 for potential substrate oxidative dehalogenation.     

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.     

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.     

Genome Mining of the Biosynthetic Gene Cluster of the Polyene Macrolide Antibiotic Tetramycin and Characterization of a P450 Monooxygenase Involved in the Hydroxylation of the Tetramycin B Polyol Segment

A polyene macrolide antibiotic tetramycin biosynthetic gene cluster was identified by genome mining and isolated from Streptomyces hygrospinosus var. beijingensis. Genetic and in silico analyses gave insights into the mechanism of biosynthesis of tetramycin, and a model of the tetramycin biosynthetic pathway is proposed. Inactivation of a cytochrome P450 monooxygenase gene, tetrK, resulted in the production of a tetramycin B precursor: tetramycin A, which lacks a hydroxy group in its polyol region. TetrK was subsequently overexpressed heterologously in E. coli with a His₆ tag, and purified TetrK efficiently hydroxylated tetramycin A to afford tetramycin B. Kinetic studies revealed no inhibition of TetrK by substrate or product. Surprisingly, sequence‐alignment analysis showed that TetrK, as a hydroxylase, has much higher homology with epoxidase PimD than with hydroxylases NysL and AmphL. The 3D structure of TetrK was then constructed by homology modeling with PimD as reference. Although TetrK and PimD catalyzed different chemical reactions, homology modeling indicated that they might share the same catalytic sites, despite also possessing some different sites correlated with substrate binding and substrate specificity. These findings offer good prospects for the production of improved antifungal polyene analogues.     

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.     

Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.