Production of Mono-Hydroxylated Derivatives of Terpinen-4-ol by Bacterial CYP102A1 Enzymes.

CYP102A1 from Bacillus megaterium is an important enzyme in biotechnology, because engineered CYP102A1 enzymes can react with diverse substrates and produce human cytochrome P450-like metabolites. Therefore, CYP102A1 can be applied to drug metabolite production. Terpinen-4-ol is a cyclic monoterpene and the primary component of essential tea tree oil. Terpinen-4-ol was known for therapeutic effects, including antibacterial, antifungal, antiviral, and anti-inflammatory. Because terpenes are natural compounds, examining novel terpenes and investigating the therapeutic effects of terpenes represent responses to social demands for eco-friendly compounds. In this study, we investigated the catalytic activity of engineered CYP102A1 on terpinen-4-ol. Among CYP102A1 mutants tested here, the R47L/F81I/F87V/E143G/L188Q/N213S/E267V mutant showed the highest activity to terpinen-4-ol. Two major metabolites of terpinen-4-ol were generated by engineered CYP102A1. Characterization of major metabolites was confirmed by liquid chromatography-mass spectrometry (LC-MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopy (NMR). Based on the LC-MS results, the difference in mass-to-charge ratio of an ion (m/z) between terpinen-4-ol and its major metabolites was 16. One major metabolite was defined as 1,4-dihydroxy-p-menth-2-ene by NMR. Given these results, we speculate that another major metabolite is also a mono-hydroxylated product. Taken together, we suggest that CYP102A1 can be applied to make novel terpene derivatives.

Production of an O-desmethylated product, a major human metabolite, of rabeprazole sulfide by bacterial P450 enzymes.

Rabeprazole is a common type of proton pump inhibitor (PPI) used to treat various peptic disorders. Unlike most PPI drugs, rabeprazole is spontaneously reduced to rabeprazole sulfide (thioether) when it is given to patients. As a result, rabeprazole sulfide is considered one of the active metabolites of rabeprazole. Rabeprazole sulfide is mainly metabolized to desmethyl rabeprazole sulfide by CYP2C19 and CYP2D6 in people. However, the pharmacological efficacy and safety of desmethyl rabeprazole sulfide have not yet been investigated. Its usage is challenging due to the high cost associated with the drug. In this study, we found CYP102A1 mutants that can produce desmethyl rabeprazole sulfide as a major metabolite of rabeprazole sulfide. The chemical characteristics of the major product were confirmed using high-performance liquid chromatography, LC-mass spectrometry, and nuclear magnetic resonance spectroscopy. CYP102A1 mutants R47L/F87V/L188Q, R47L/F87V/L188Q/A335V/Q359R, and R47L/F87V/L188Q/I254V/D351E showed kcat values of 39, 93, and 88 min-1, respectively, for O-desmethylation of rabeprazole sulfide. Furthermore, the highest concentration of desmethyl rabeprazole sulfide product from 2 mM rabeprazole sulfide at optimal conditions was obtained in bacterial whole-cell biotransformation with the R47L/F87V/L188Q mutant, reaching 0.63 mM at 4-h incubation. In conclusion, we present a platform that facilitates the efficient and sustainable production of the desmethylated product from rabeprazole sulfide for use in the biopharmaceutical industry.

CYP102A1 peroxygenase catalyzed reaction via in situ H2O2 generation.

CYP102A1 is a promiscuous bacterial cytochrome P450 (CYP or P450) known for its diverse substrates and comparable activity with human P450 enzymes. The development of CYP102A1 peroxygenase activity can contribute significantly to human drug development and drug metabolite production. Peroxygenase has recently emerged as an alternative to a dependency of P450 on NADPH-P450 reductase and NADPH cofactor and gives more opportunity for practical application. However, the H2O2 dependency also leads to challenges regarding its practical application, in which the excessive H2O2 concentration causes the activation of the peroxygenases. Therefore, we need the optimization of H2O2 production to minimize oxidative inactivation. In this study, we report the CYP102A1 peroxygenase-catalyzed atorvastatin hydroxylation reaction with an enzymatic H2O2 generation using glucose oxidase. Random mutagenesis at the CYP102A1 heme domain was used to generate mutant libraries with high throughput screening of highly active mutants, which can pair with the in situ H2O2 generation. The setup of the CYP102A1 peroxygenase reaction was also possible for other statin drugs and could be developed to produce drug metabolites. We also found a relationship between enzyme inactivation and product formation during the catalytic reaction, supported by enzymatic in situ H2O2 supply. It can be suggested that the low product formation is due to enzyme inactivation.

Production of a major metabolite of niclosamide using bacterial cytochrome P450 enzymes.

Niclosamide has been proposed as a possible candidate for a Covid-19 drug. However, the metabolites of niclosamide are difficult to investigate because they are usually not available commercially or they are quite expensive in the commercial market. In this study, the major metabolite of niclosamide in human liver microsomes (HLMs) was confirmed to be 3-OH niclosamide. Because the production of 3-OH niclosamide using HLMs has a slow turnover rate, a new method of producing niclosamide metabolite with an easier and highly cost-efficient method was thus conducted. Bacterial CYP102A1 (BM3) is one of the bacterial cytochrome P450s (CYPs) from Bacillus megaterium that structurally show similar activities to human CYPs. Here, the BM3 mutants were used to produce niclosamide metabolites and the metabolites were analyzed using high-performance liquid chromatography and LC-mass spectrometry. Among a set of mutants tested here, BM3 M14 mutant was the most active in producing 3-OH niclosamide, the major metabolite of niclosamide. Comparing BM3 M14 and HLMs, BM3 M14 production of 3-OH niclosamide was 34-fold higher than that of HLMs. Hence, the engineering of BM3 can be a cost-efficient method to produce 3-OH niclosamide.

Production of 3,4-dihydroxy-L-phenylalanine using novel tyrosinases from Bacillus megaterium.

Tyrosinases, type-3 copper proteins responsible for melanin formation in various organisms, have considerable potential to produce bioactive catechol derivatives such as 3,4-dihydroxy-L-phenylalanine (L-DOPA). They catalyze the ortho-hydroxylation of L-tyrosine to L-DOPA via monophenolase activity and the subsequent oxidation of L-DOPA to dopaquinone through diphenolase activity, which then spontaneously converts to melanin. In this study, six novel Bacillus megaterium strains, GJ802, GJ803, DY801, DY802, DY804, and DY805, were isolated from rice straw in South Korea. The tyrosinases of the novel strains were cloned, purified, and characterized. They exhibited catalytic activity over a broad pH range and showed high thermal stability. In addition, a tyrosinase of the B. megaterium DY805 strain (DY805), having the highest monophenolase activity among the tyrosinases, was used to produce L-DOPA as a biocatalyst. DY805 produced 8.77 mg/L L-DOPA from 200 µM L-tyrosine (36.2 mg/L), with a yield of 23.3%. After the optimization of several parameters for L-DOPA production, DY805 could produce up to 264 mg/L L-DOPA (30-fold increase), with a yield of 97.2% from 1500 µM L-tyrosine (272 mg/L). Taken together, these novel tyrosinases could be considered useful biocatalysts in L-DOPA production and other biotechnology fields.

Assessment of standardization of domestic commercial black garlic extract for S-allyl-l-cysteine and S-1-propenyl-l-cysteine.

Aged garlic extract (AGE) contains a significant amount of bioactive compounds, including S-allyl-l-cysteine (SAC), which is associated with various health benefits. Among different AGE products, black garlic extract (BGE) is widely consumed and a common product in the Korean market. BGE products do however contain different levels of SAC and S1PC. Here, the SAC contents in commercial BGE products were found to be in the range of 0.31-27.22 mg/100 mL, while the SAC contents of commercial black garlic (BG) cloves were in the range of 22.28-63.71 mg/100 g. Recently, S-1-propenyl-l-cysteine (S1PC) has emerged as a new bioactive compound of interest in AGE products. Analysis of BG and BGE indicated that their S1PC contents were 2.24-16.58 mg/100 g and ND-3.68 mg/100 mL, respectively. Based on the significance of these compounds, standardization of the SAC and S1PC content in commercial BGE products is required.

Roles of Human Liver Cytochrome P450 Enzymes in Tenatoprazole Metabolism.

Tenatoprazole, a newly developed proton pump inhibitor candidate, was developed as an acid inhibitor for gastric acid hypersecretion disorders such as gastric ulcer and reflux esophagitis. It is known that tenatoprazole is metabolized to three major metabolites of 5'-hydroxy tenatoprazole, tenatoprazole sulfide, and tenatoprazole sulfone in human liver, primarily catalyzed by CYPs 2C19 and 3A4. While CYP2C19 prefers the hydroxylation of tenatoprazole at C-5' position, CYP3A4 is mainly involved in sulfoxidation reaction to make tenatoprazole sulfone. Tenatoprazole sulfide is a major human metabolite of tenatoprazole and is formed spontaneously and non-enzymatically from tenatoprazole. However, its metabolic fate in the human liver is not fully known. Furthermore, no systematic metabolic study has been performed to study tenatoprazole or tenatoprazole sulfide. Here, we studied the functions of human cytochromes P450 in the metabolic pathway of tenatoprazole and tenatoprazole sulfide by using recombinant human P450s and human liver microsomes. Both CYP 2C19 and CYP3A4 showed distinct regioselective and stereospecific monooxygenation activities toward tenatoprazole and tenatoprazole sulfide. Furthermore, a new major metabolite of tenatoprazole sulfide was found, 1'-N-oxy-5'-hydroxytenatoprzole sulfide, which has never been reported. In conclusion, the metabolic fates of tenatoprazole and tenatoprazole sulfide should be considered in the clinical use of tenatoprazole.

A Novel Statin Compound from Monacolin J Produced Using CYP102A1-Catalyzed Regioselective C-Hydroxylation.

Statins inhibit the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), which is the rate-limiting enzyme in cholesterol biosynthesis. Statin therapy reduces morbidity and mortality in those who are at high risk of cardiovascular disease. Monacolin J is a statin compound, which is an intermediate in the lovastatin biosynthesis pathway, in the fungus Aspergillus terreus. It is also found in red yeast rice, which is made by culturing rice with the yeast Monascus purpureus. Monacolin J has a hydroxyl substituent at position C'-8 of monacolin L. Here, a new statin derivative from monacolin J was made through the catalysis of CYP102A1 from Bacillus megaterium. A set of CYP102A1 mutants of monacolin J hydroxylation with high catalytic activity was screened. The major hydroxylated product was C-6'a-hydroxymethyl monacolin J, whose structure was confirmed using LC-MS and NMR analysis. The C-6'a-hydroxymethyl monacolin J has never been reported before. It showed a greater ability to inhibit HMG-CoA reductase than the monacolin J substrate itself. Human liver microsomes and human CYP3A4 also showed the ability to catalyze monacolin J in producing the same product of the CYP102A1-catalyzed reaction. This result motivates a new strategy for the development of a lead for the enzymatic and chemical processes to develop statin drug candidates.

Enzymatic Production of 3-OH Phlorizin, a Possible Bioactive Polyphenol from Apples, by Bacillus megaterium CYP102A1 via Regioselective Hydroxylation.

Phlorizin is the most abundant glucoside of phloretin from the apple tree and its products. Phlorizin and its aglycone phloretin are currently considered health-beneficial polyphenols from apples useful in treating hyperglycemia and obesity. Recently, we showed that phloretin could be regioselectively hydroxylated to make 3-OH phloretin by Bacillus megaterium CYP102A1 and human P450 enzymes. The 3-OH phloretin has a potent inhibitory effect on differentiating 3T3-L1 preadipocytes into adipocytes and lipid accumulation. The glucoside of 3-OH phloretin would be a promising agent with increased bioavailability and water solubility compared with its aglycone. However, procedures to make 3-OH phlorizin, a glucoside of 3-OH phloretin, using chemical methods, are not currently available. Here, a biocatalytic strategy for the efficient synthesis of a possibly valuable hydroxylated product, 3-OH phlorizin, was developed via CYP102A1-catalyzed regioselective hydroxylation. The production of 3-OH phlorizin by CYP102A1 was confirmed by HPLC and LC-MS spectroscopy in addition to enzymatic removal of its glucose moiety for comparison to 3-OH phloretin. Taken together, in this study, we found a panel of mutants from B. megaterium CYP102A1 could catalyze regioselective hydroxylation of phlorizin to produce 3-OH phlorizin, a catechol product.

Biotransformation of Flavonoids by Newly Isolated and Characterized Lactobacillus pentosus NGI01 Strain from Kimchi.

Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) microorganisms. This study aimed to identify novel LAB strains that can transform flavonoids into aglycones to improve bioavailability. We isolated 34 LAB strains from kimchi. The biotransformation activity of these 34 LAB strains was investigated based on α-L-rhamnosidase and ß-D-glucosidase activities. Among them, 10 LAB strains with high activities were identified by 16S rRNA sequencing analysis. All tested LAB strains converted hesperidin to hesperetin (12.5-30.3%). Of these, only the Lactobacillus pentosus NGI01 strain produced quercetin from rutin (3.9%). The optimal biotransformation conditions for the L. pentosus NGI01 producing hesperetin and quercetin were investigated. The highest final product concentrations of hesperetin and quercetin were 207 and 78 µM, respectively. Thus, the L. pentosus NGI01 strain can be a biocatalyst for producing flavonoid aglycones in the chemical and food industries.

Regioselective Hydroxylation of Phloretin, a Bioactive Compound from Apples, by Human Cytochrome P450 Enzymes.

Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min-1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h-1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6ß-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.

Biochemical analysis of recombinant CYP4A11 allelic variant enzymes: W126R, K276T and S353G.

Human CYP4A11 is the major ω-hydroxylase of fatty acids in the liver and kidneys. It produces 20-hydroxyeicosatetraenoic acid as well as hydroxylates fatty acids. In this study, we investigated the biochemical properties of three alleles of CYP4A11: W126R, K276T, and S353G. Site-directed mutagenesis of the wild type CYP4A11 was performed, to construct the W126R, K276T, and S353G variant clones. The CYP4A11 wild type and variant constructs were heterologously expressed in Escherichia coli. CO-binding spectra showed the expression of the wild type, K276T and S353G variants, indicating the functional P450 holoenzyme. The W126R variant was not expressed in E. coli. Binding affinities of lauric acid in K276T and S353G variants were stronger than that of wild type. Steady-state kinetics in the hydroxylation reaction of fatty acids were studied. The catalytic efficiencies (kcat/Km) of K276T and S353G variants in the reactions without cytochrome b5 were approximately 2- and 4-fold higher, respectively, than that of wild type, and in the reactions with cytochrome b5 they were approximately 2- and 3-fold higher, respectively. These results suggest that individuals carrying the alleles, K276T and S353G, might exhibit higher catalysis of CYP4A11, which may affect the endogenous metabolic products associated with regulation of blood pressure.

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue.

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A.

Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 µM and 2.0 ± 0.1 µM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 µM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.

Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. Here, we have designed a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17ß-estradiol), to demonstrate the general applicability of the light-driven, cofactor-free system.

Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.

Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.

Regioselective hydroxylation of 17ß-estradiol by mutants of CYP102A1 from Bacillus megaterium.

A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17ß-estradiol (E2). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E2 in this study. Some of the tested mutants could regioselectively generate 2-OH E2 as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.

Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.

Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.