P450 BM3 Research Articles

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.

MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels.

Microgels are an emerging class of "ideal" enzyme carriers because of their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly(N-vinylcaprolactam) (PVCL) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as a comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. P450 monooxygenases are usually challenging to immobilize, and often, high activity losses occur after the immobilization (in the case of P450 BM3 from Bacillus megaterium up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels was achieved without the loss of catalytic activity at the pH optimum of P450 BM3 (pH 8; ∼9.4 nmol 7-hydroxy-3-carboxy coumarin ethyl ester/min for free and immobilized P450 BM3); the resulting P450-microgel systems were termed P450 MicroGelzymes (P450 μ-Gelzymes). In addition, P450 μ-Gelzymes offer the possibility of reversible ionic strength-triggered release and re-entrapment of the biocatalyst in processes (e.g., for catalyst reuse). Finally, a characterization of the potential of P450 μ-Gelzymes to provide resistance against cosolvents (acetonitrile, dimethyl sulfoxide, and 2-propanol) was performed to evaluate the biocatalytic application potential.

Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins.

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA-CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174µM), a similar amount when compared to the fully functional fusion protein (176µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA-CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.

Mechanism of deep eutectic solvents enhancing catalytic function of cytochrome P450 enzymes in biosynthesis and organic synthesis

Indigo is an insoluble blue dye, which generates serious pollution in its production process. Increasing focus has come to the biosynthesis of indigo that are more environment-preserved and high-efficient. Hence, this study was designed to explore the specific role of various deep eutectic solvents (DESs) on cytochromeP45-BM-3 catalyzing indole to produce indigo. DESs were synthesized by heating and stirring. The structure of the solvent was analyzed by nuclear magnetic resonance (NMR) and fourier transform infrared spectrum (FT-IR), and the relationship between the viscosity, density and refractive index of the solvent, and the water content of the solvent was examined. Circular dichroism spectrometer was used to detect the tertiary structure of the enzyme protein. The effect of solvent type, concentration, pH, temperature, and water content on the catalytic activity and stability of P450 BM-3 was measured using an ultraviolet spectrophotometer. A new solvent biphasic system was established using DESs and buffers, and indigo was prepared using recombinant E. coli-biocatalyzed indole. DESs were low-melting eutectics formed by molecules interaction of components through hydrogen bonding. The physical properties of DESs such as density, viscosity, and refractive index varied with water content and temperature of the solvent. The pH, water content, and temperature of DESs were positively correlated with the catalytic activity of P450 BM-3. To sum up, DESs can improve the catalytic activity and thermal stability of P450 BM-3. Indigo can be efficiently prepared using the DESs-buffer biphasic system.

Promoting P450 BM3 heme domain dimerization with a tris(5-iodoacetamido-1,10-phenanthroline)Ru(II) complex.

Protein dimerization often occurs in many biological systems as to provide structural and functional advantages. A tris(5-iodoacetamido-1,10-phenanthroline)Ruthenium(II) complex was shown to promote the covalent dimerization of a P450 BM3 heme domain mutant containing a surface exposed non-native single cysteine residue. The formation of homodimeric species was confirmed by protein gel electrophoresis, mass spectrometry and UV-Vis spectroscopy. The dimeric species could be separated from the monomer and aggregates by size-exclusion chromatography. Docking simulation reveals a plausible structure with two proteins covalently conjugated to the inorganic compound.

Surface charge-controlled electron transfer and catalytic behavior of immobilized cytochrome P450 BM3 inside dendritic mesoporous silica nanoparticles.

Understanding the influencing factors on the reaction kinetics of P450 BM3 within confined spaces is essential for developing efficient P450 BM3 bioreactors. Herein, two dendritic mesoporous silica nanoparticles (OH-DMSNs and NH2-DMSNs) with similar pore size but opposite surface charge have been prepared and served as the vehicle to immobilize P450 BM3. With the help of the film-forming material of chitosan, P450 BM3/OH-DMSN and P450 BM3/NH2-DMSN composites were immobilized on GC electrode and characterized with electrochemical measurements. Compared with P450 BM3/OH-DMSNs/GCE, P450 BM3/NH2-DMSNs/GCE showed higher electron transfer efficiency with higher current charge and lower ks value. Besides, the generated catalytic current towards testosterone on P450 BM3/NH2-DMSNs/GCE was 1.81 times larger than P450 BM3/OH-DMSNs/GCE. Furthermore, P450 BM3 inside NH2-DMSNs displayed higher affinity towards testosterone with the lower Kmapp value of 244.82μM. These results are attributed to the positively charged internal walls of NH2-DMSNs so that P450 BM3 adapts to an orientation favorable for electron exchange with electrodes and substrate binding with the active sites. The present study provides fundamentals for regulating the surface charge to optimize redox process and catalytic behavior in CYP bioreactors through electrostatic interactions.

Process intensification for cytochrome P450 BM3-catalyzed oxy-functionalization of dodecanoic acid.

Selective oxy‐functionalization of nonactivated C‐H bonds is a long‐standing “dream reaction” of organic synthesis for which chemical methodology is not well developed. Mono‐oxygenase enzymes are promising catalysts for such oxy‐functionalization to establish. Limitation on their applicability arises from low reaction output. Here, we showed an integrated approach of process engineering to the intensification of the cytochrome P450 BM3‐catalyzed hydroxylation of dodecanoic acid (C12:0). Using P450 BM3 together with glucose dehydrogenase for regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), we compared soluble and co‐immobilized enzymes in O2‐gassed and pH‐controlled conversions at high final substrate concentrations (≥40mM). We identified the main engineering parameters of process output (i.e., O2 supply; mixing correlated with immobilized enzyme stability; foam control correlated with product isolation; substrate solubilization) and succeeded in disentangling their complex interrelationship for systematic process optimization. Running the reaction at O2‐limited conditions at up to 500‐ml scale (10% dimethyl sulfoxide; silicone antifoam), we developed a substrate feeding strategy based on O2 feedback control. Thus, we achieved high reaction rates of 1.86g·L−1·hr−1 and near complete conversion (≥90%) of 80mM (16g/L) C12:0 with good selectivity (≤5% overoxidation). We showed that “uncoupled reaction” of the P450 BM3 (~95% utilization of NADPH and O2 not leading to hydroxylation) with the C12:0 hydroxylated product limited the process efficiency at high product concentration. Hydroxylated product (~7g; ≥92% purity) was recovered from 500ml reaction in 82% yield using ethyl‐acetate extraction. Collectively, these results demonstrate key engineering parameters for the biocatalytic oxy‐functionalization and show their integration into a coherent strategy for process intensification.

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

Microbial enzyme stereoselectively converts EPA into 17(S),18(R)-epoxyeicosatetraenoic acid useful for the amelioration of contact hypersensitivity

Abstract Dietary intake of ω3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid are beneficial for health control because ω3 fatty acids are metabolized to pro-resolution and anti-inflammatory lipid metabolites. We previously identified 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) as a new lipid metabolite endogenously generated from EPA that exhibits potent anti-allergic and anti-inflammatory properties. However, the chemically synthesized 17,18-EpETE is enantiomeric mixture concerning to its epoxy group, i.e., 17(S),18(R)-EpETE and 17(R),18(S)-EpETE, and the stereostructural differences in anti-inflammatory effect of 17,18-EpETE has not been clarified. Because it is necessary to identify which isomer shows physiological function to develop it as a medicine, we evaluated stereospecific effects of 17(S),18(R)-EpETE and 17(R),18(S)-EpETE in amelioration of skin contact hypersensitivity. As a result, we found that anti-inflammatory activity was detected in 17(S),18(R)-EpETE, but not 17(R),18(S)-EpETE. In addition, we found that cytochrome P450 BM-3 derived from Bacillus megaterium stereoselectively converts EPA into 17(S),18(R)-EpETE, which effectively inhibited the development of contact hypersensitivity by inhibiting neutrophil migration in a G protein-coupled receptor 40-dependent manner. These results demonstrated that immune cells (e.g., neutrophils) distinguish biological active lipid metabolites stereoselectively for the control of their activity, which is a new strategy for the development of efficient immunotherapy.

Full Structure Modeling of Three-Domains Monooxygenase CYP102A1 ВМ3 from Bacillus megaterium

The 3D full-atom model of the whole-size CYP102A1 from Bacillus megaterium (cytochrome P450 BM3) has been constructed using molecular modeling methods. The structure model was constructed using crystal structures of the separate FAD-binding domain (PDB ID: 4DQK) and the complex of FMN-binding and monooxygenase domains (PDB ID: 1BVY). Modeling procedure included analysis of the domains’ surfaces to find the orientation with maximum inter-subunit contacts. The overall configuration of the obtained complex was optimized using molecular dynamics. The final full-atom structure model shows rather tight interactions between FAD- and FMN-binding domains due to 10 inter-domain hydrogen bonds and hydrophobic interactions between three pairs of amino acid residues. This 3D model can be used for structure-function studies and rational design of the enzyme as well as for construction of hybrid supramolecular structures of biocatalysts with cytochrome P450 BM3.

Surface Display of Complex Enzymes by in Situ SpyCatcher-SpyTag Interaction.

The display of complex proteins on the surface of cells is of great importance for protein engineering and other fields of biotechnology. Herein, we describe a modular approach, in which the membrane anchor protein Lpp‐OmpA and a protein of interest (passenger) are expressed independently as genetically fused SpyCatcher and SpyTag units and assembled in situ by post‐translational coupling. Using fluorescent proteins, we first demonstrate that this strategy allows the construct to be installed on the surface of E. coli cells. The scope of our approach was then demonstrated by using three different functional enzymes, the stereoselective ketoreductase Gre2p, the homotetrameric glucose 1‐dehydrogenase GDH, and the bulky heme‐ and diflavin‐containing cytochrome P450 BM3 (BM3). In all cases, the SpyCatcher‐SpyTag method enabled the generation of functional whole‐cell biocatalysts, even for the bulky BM3, which could not be displayed by conventional fusion with Lpp‐OmpA. Furthermore, by using a GDH variant carrying an internal SpyTag, the system could be used to display an enzyme with unmodified N‐ and C‐termini.

Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells

Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

17(S),18(R)-epoxyeicosatetraenoic acid generated by cytochrome P450 BM-3 from Bacillus megaterium inhibits the development of contact hypersensitivity via G-protein-coupled receptor 40-mediated neutrophil suppression.

Dietary intake of ω3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid is beneficial for health control. We recently identified 17,18‐epoxyeicosatetraenoic acid (17,18‐EpETE) as a lipid metabolite endogenously generated from eicosapentaenoic acid that exhibits potent anti‐allergic and anti‐inflammatory properties. However, chemically synthesized 17,18‐EpETE is enantiomeric due to its epoxy group—17(S),18(R)‐EpETE and 17(R),18(S)‐EpETE. In this study, we demonstrated stereoselective differences of 17(S),18(R)‐EpETE and 17(R),18(S)‐EpETE in amelioration of skin contact hypersensitivity and found that anti‐inflammatory activity was detected in 17(S),18(R)‐EpETE, but not in 17(R),18(S)‐EpETE. In addition, we found that cytochrome P450 BM‐3 derived from Bacillus megaterium stereoselectively converts EPA into 17(S),18(R)‐EpETE, which effectively inhibited the development of skin contact hypersensitivity by inhibiting neutrophil migration in a G protein‐coupled receptor 40‐dependent manner. These results suggest the new availability of a bacterial enzyme to produce a beneficial lipid mediator, 17(S),18(R)‐EpETE, in a stereoselective manner. Our findings highlight that bacterial enzymatic conversion of fatty acid is a promising strategy for mass production of bioactive lipid metabolites.

Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3

AbstractNatural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4‐phenyl‐2‐butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active‐site variants of P450 BM3 using orthogonal high‐throughput workflows to identify the most streamlined route to all indigo‐forming variants. Among the three known and 13 new indigo‐forming variants, eight hydroxylated 4‐phenyl‐2‐butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3.

An integrated screening system for the selection of exemplary substrates for natural and engineered cytochrome P450s

Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

Selective Functionalization of Microgels with Enzymes by Sortagging.

Enzyme immobilization has been widely used to improve the stability and recyclability of enzymes in industrial processes. In this work, a sortase-mediated and therefore selective covalent immobilization strategy (sortagging) for enzymes on microgels (GelZyms) was investigated. Aqueous microgels were synthesized from poly(N-vinylcaprolactam)/glycidyl methacrylate (PVCL/GMA) and tagged with the sortase A recognition peptide sequence (LPETG) or its nucleophilic counterpart-tag (GGG). General applicability and selective immobilization were confirmed by subsequent sortagging of five different enzymes (Bacillus subtilis lipase A (BSLA), Yersinia mollaretii phytase (Ym-phytase), Escherichia coli copper efflux oxidase (CueO laccase), cellulase A2, and Bacillus megaterium monooxygenase P450 BM3). The latter was performed directly from the cell lysate to ensure cost-effective immobilization. All five immobilized enzymes were catalytically active and could be recycled (e.g., laccase CueO and monooxygenase P450 BM3 F87A; >55% residual activity after six cycles). Application potential was demonstrated by using CueO decorated microgels for bleaching of the synthetic dye indigo carmine.

One‐Pot Two‐Step Chemoenzymatic Cascade for the Synthesis of a Bis‐benzofuran Derivative

Chemoenzymatic cascades enable reactions with the high productivity of chemocatalysts and high selectivity of enzymes. Nevertheless, the combination of these different fields of catalysis is prone to mutual deactivation of metal‐ and biocatalysts. In this study, a one‐pot sequential two‐step catalytic cascade reaction was successfully implemented for the synthesis of a methylene‐bridged bis(2‐substituted benzofuran). In the first step, a palladium‐free Sonogashira reaction is used for the synthesis of a benzofuran derivative. In the subsequent step, the formed 2‐substituted benzofuran is hydroxylated by the monooxygenase P450 BM3 variant (A74S‐F87V‐L188Q) and undergoes further elimination reactions. The study proofs that combination of Cu scorpionate catalyzed Sonogashira cross‐coupling and P450 mediated oxidation is possible and results in up to 84 % yield of the final product. The oxidation reaction is boosted by capturing inhibiting reaction components.

Enzymatic cascade biosynthesis reaction of musky macrolactones from fatty acids

Musky macrolactones are an important group of compounds used in high-valued perfumery. An enzymatic cascade reaction including cytochrome P450 hydroxylase and lipase was explored to biosynthesize musky macrolactones. Firstly, fatty acids were hydroxylated by P450 hydroxylase to produce the corresponding ω-hydroxy fatty acids. Then ω-hydroxy fatty acids were lactonized by lipase. ω-Hydroxy fatty acids can difficultly be synthesized by traditional chemical methods, and the production of these compounds were key constraint factors during the whole reaction. To obtain enough precursors of macrolactones, an efficient production of ω-hydroxy fatty acids was explored. A mutant of P450 BM3 from Bacillus megaterium was used as terminal hydroxylases. To improve the yield of ω-hydroxy fatty acids, the coenzyme regeneration system and auxiliary organic solvent were optimized. The conversion using the P450 BM3 mutant under the biphase system was up to 42% towards ω-hydroxy pentadecanoic acid and 98% towards ω-hydroxy palmitic acid. The results reveal that the musky macrolactones, exaltolide and silvanone supra, could be synthesized in the hydroxylation-lactonization cascade reaction. Finally, 81 mg of exaltolide was obtained from 242 mg pentadecanoic acid, and 199 mg of silvanone supra from 256 mg palmitic acid.

The Registration of a Biomaser-Like Effect in an Enzyme System with an RTM Sensor

The concentration dependence of a microwave frequency radiation from a solution of a functioning enzyme system (ES) (with the example of cytochrome P450 BM3 (CYP102A1) system during lauric acid (LA) hydroxylation) has been studied with a radiothermometric sensor. Registration of the radiation from the enzyme solution has been performed in the frequency range from 3.4 to 4.2 GHz at the enzyme concentrations from 10−10М to 10−6М. It has been demonstrated that the catalysis of LA hydroxylation in a reconstituted CYP102A1 system is accompanied by a generation of microwave radiation over the entire range of concentrations studied. It has been found that a transition from a multipulse mode (at nanomolar enzyme concentrations from 10−10М to 10−8М) to a single-pulse mode (at micromolar enzyme concentrations from 10−7М to 10−6М) is observed. This effect is discussed on the basis of assumptions considering possible realization of biomaser-like radiation in the enzyme system. The discovered concentration-based effect of the transition of an unsynchronized pulsed radiation into a synchronized one in ES can further be used in the development of novel methods of noninvasive diagnostics of diseases, in mathematical modeling of the functioning of living systems, and in the development of next-generation quantum computers.