Oxidation of Naringenin, Apigenin, and Genistein by Human Family 1 Cytochrome P450 Enzymes and Comparison of Interaction of Apigenin with Human P450 1B1.1 and Scutellaria P450 82D.1.

Naringenin, an initial synthesized flavanone in various plant species, is further utilized for production of many biologically active flavonoids, e.g., apigenin, eriodictyol, and genistein, by various plant enzymes including cytochrome P450s (P450s or CYPs). We examined how these flavonoids are oxidized by human P450 family 1 and 2A enzymes. Naringenin was principally oxidized at the 3'-position to form eriodictyol by CYP1 enzymes more efficiently than by CYP2A enzymes, and the resulting eriodictyol was further oxidized to two penta-hydroxylated products. In contrast to plant P450 enzymes, these human P450s did not mediate the desaturation of naringenin and eriodictyol to give apigenin and luteolin, respectively. Apigenin was oxidized at the C3' and C6 positions to form luteolin and scutellarein by these P450s. CYP1B1.1 and 1B1.3 had high activities in apigenin 6-hydroxylation with a homotropic cooperative manner, as has been observed previously in chrysin 6-hydroxylation (Nagayoshi et al., Chem. Res. Toxicol. 2019, 32, 1268-1280). Molecular docking analysis suggested that CYP1B1 had two apigenin binding sites and showed similarities in substrate recognition sites to plant CYP82D.1, one of the enzymes in catalyzing apigenin and chrysin 6-hydroxylations in Scutellaria baicalensis. The present results suggest that human CYP1 enzymes and CYP2A13 in some reactions have important roles in the oxidation of naringenin, eriodictyol, apigenin, and genistein and that human CYP1B1 and Scutellaria CYP82D.1 have similarities in their SRS regions, catalyzing 6-hydroxylation of both apigenin and chrysin.

Functional Characterization of Allelic Variations of Human Cytochrome P450 2C8 (V181I, I244V, I331T, and L361F).

The human cytochrome P450 2C8 is responsible for the metabolism of various clinical drugs as well as endogenous fatty acids. Allelic variations can significantly influence the metabolic outcomes. In this study, we characterize the functional effects of four nonsynonymous single nucleotide polymorphisms *15, *16, *17, and *18 alleles recently identified in cytochrome P450 2C8. The recombinant allelic variant enzymes V181I, I244V, I331T, and L361F were successfully expressed in Escherichia coli and purified. The steady-state kinetic analysis of paclitaxel 6-hydroxylation revealed a significant reduction in the catalytic activities of the V181I, I244V, and L361F variants. The calculated catalytic efficiency (kcat/Km) of these variants was 5-26% of that of the wild-type enzyme. The reduced activities were due to both decreased kcat values and increased Km values of the variants. The epoxidation of arachidonic acid by the variants was analyzed. The L361F variant only exhibited 4-6% of the wild-type catalytic efficiency in ω-9- and ω-6-epoxidation reactions to produce 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, respectively. These reductions were mainly due to a decrease in the kcat value of the L361F variant. The binding titration analysis of paclitaxel and arachidonic acid showed that all variants had similar affinities to those of the wild-type (10-14 µM for paclitaxel and 20-49 µM for arachidonic acid). The constructed paclitaxel docking model of the variant enzyme suggests that the L361F substitution leads to the incorrect orientation of paclitaxel in the active site, with the 6'C of paclitaxel displaced from the productive catalytic location. This study suggests that individuals carrying the newly identified P450 2C8 allelic variations are likely to have an altered metabolism of clinical medicines and production of fatty acid-derived signal molecules.

Tight binding of cytochrome b5 to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis.

It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5-P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b5 that can be used in titrations. The labeled b5 bound to WT P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s-1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Structural characterization and fatty acid epoxidation of CYP184A1 from Streptomyces avermitilis.

The genome of Streptomyces avermitilis contains 33 cytochrome P450 genes. Among the P450 gene products of S. avermitilis, we characterized the biochemical function and structural aspects of CYP184A1. Ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that CYP184A1 induced an epoxidation reaction to produce 9,10-epoxystearic acid. Steady-state kinetic analysis yielded a kcat value of 0.0067 min-1 and a Km value 10 µM. The analysis of its crystal structures illustrated that the overall CYP184A1 structure adopts the canonical scaffold of cytochrome P450 and possesses a narrow and deep substrate pocket architecture that is required for binding to linear chain fatty acids. In the structure of the CYP184A1 oleic acid complex (CYP184A1-OA), C9-C10 of oleic acid was bound to heme for the productive epoxidation reaction. This study elucidates the roles of P450 enzymes in the oxidative metabolism of fatty acids in Streptomyces species.

Oxidation of 3'-methoxyflavone, 4'-methoxyflavone, and 3',4'-dimethoxyflavone and their derivatives having 5,7-dihydroxyl moieties by human cytochromes P450 1B1 and 2A13.

Oxidation of 3'-methoxyflavone, 4'-methoxyflavone, and 3',4'-dimethoxyflavone and their derivatives containing 5,7-dihydroxyl groups by human cytochrome P450 (P450 or CYP) 1B1 and 2A13 was determined using LC-MS/MS systems.3'-Methoxyflavone and 4'-methoxyflavone were mainly O-demethylated to form 3'-hydroxyflavone and 4'-hydroxyflavone, respectively, and then 3',4'-dihydroxyflavone at higher rates with CYP1B1 than with CYP2A13. 4'-Methoxy-5,7-dihydroxyflavone (acacetin) was found to be demethylated by CYP1B1 and 2A13 to form 4',5,7-trihydroxyflavone (apigenin) at rates of 0.098-1 and 0.42 min-1, respectively. 3'-Methoxy-5,7-dihydroxyflavone was also demethylated by both P450s, with CYP2A13 being more active.3',4'-Dimethoxyflavone was a good substrate for CYP1B1 but not for CYP2A13 and was found to be mainly O-demethylated to form 3',4'-dihydroxyflavone (at a rate of 4.2 min-1) and also several ring-oxygenated products having m/z 299 fragments. Molecular docking analysis supported the proper orientation for formation of these products by CYP1B1.Our present results showed that 3'- and 4'-methoxyflavone can be oxidised to their O-demethylated products and, to a lesser extent, to ring oxidation products by both P450s 1B1 and 2A13 and that 3',4'-dimethoxyflavone is a good substrate for CYP1B1 in forming both O-demethylated and ring-oxidation products. Introduction of a 57diOHF moiety into these methoxylated flavonoids caused decreased in oxidation by CYP1B1 and 2A13.

TNP and its analogs: Modulation of IP6K and CYP3A4 inhibition.

Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.

Cytochrome b 5 Binds Tightly to Several Human Cytochrome P450 Enzymes.

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5, that could be used in binding assays at sub-µM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high, and that ternary P450-b 5-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT: High-affinity binding of cytochrome b 5 (b 5) (K d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b 5-NADPH-P450 reductase complex.

Roles of cytochrome P450 2A6 in the oxidation of flavone, 4'-hydroxyflavone, and 4'-, 3'-, and 2'-methoxyflavones by human liver microsomes.

Nine forms of recombinant cytochrome P450 (P450 or CYP) enzymes were used to study roles of individual P450 enzymes in the oxidation of flavone and some other flavonoids, 4'-hydroxyflavone and 4'-, 3'-, and 2'-methoxyflavones, by human liver microsomes using LC-MS/MS analysis.As has been reported previously , 4'-, 3'-, and 2'-methoxyflavones were preferentially O-demethylated by human liver P450 enzymes to form 4'-, 3'-, and 2'-hydroxylated flavones and also 3',4'-dihydroxyflavone from the former two substrates.In comparisons of product formation by oxidation of these methoxylated flavones, CYP2A6 was found to be a major enzyme catalysing flavone 4'- and 3'-hydroxylations by human liver microsomes but did not play significant roles in 2'-hydroxylation of flavone, O-demethylations of three methoxylated flavones, and the oxidation of 4'-hydroxyflavone to 3',4'-dihydroxyflavone.The effects of anti-CYP2A6 IgG and chemical P450 inhibitors suggested that different P450 enzymes, as well as CYP2A6, catalysed oxidation of these flavonoids at different positions by liver microsomes.These studies suggest that CYP2A6 catalyses flavone 4'- and 3'-hydroxylations in human liver microsomes and that other P450 enzymes have different roles in oxidizing these flavonoids.

Liquid chromatography-tandem mass spectrometry analysis of oxidation of 2'-, 3'-, 4'- and 6-hydroxyflavanones by human cytochrome P450 enzymes.

2'-Hydroxyflavanone (2'OHFva), 3'OHFva, 4'OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3'OHFva and 4'OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4'OHFva (m/z 241) was used as a substrate, formation of 4'OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4'OHFvo by P450s. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

Structural insights into CYP107G1 from rapamycin-producing Streptomyces rapamycinicus.

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 µM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.

Preference for O-demethylation reactions in the oxidation of 2'-, 3'-, and 4'-methoxyflavones by human cytochrome P450 enzymes.

2'-, 3'-, and 4'-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products formed were analyzed with LC-MS/MS methods.CYP1B1.1 and 1B1.3 were highly active in demethylating 4'MeF to form 4'-hydroxyflavone (rate of 5.0 nmol/min/nmol P450) and further to 3',4'-dihydroxyflavone (rates of 2.1 and 0.66 nmol/min/nmol P450, respectively). 3'MeF was found to be oxidized by P450s to m/z 239 (M-14) products (presumably 3'-hydroxyflavone) and then to 3',4'-dihydroxyflavone. P450s also catalyzed oxidation of 2'MeF to m/z 239 (M-14) and m/z 255 (M-14, M-14 + 16) products, presumably mono- and di-hydroxylated products, respectively.At least two types of ring oxidation products having m/z 269 fragments were formed, although at slower rates than the formation of mono- and di-hydroxylated products, on incubation of these MeFs with P450s; one type was products oxidized at the C-ring, having m/z 121 fragments, and the other one was the products oxidized at the A-ring (having m/z 137 fragments).Molecular docking analysis indicated the preference of interaction of O-methoxy moiety of methoxyflavones in the active site of CYP1A2.These results suggest that 2'-, 3'-, and 4'-methoxyflavones are principally demethylated by human P450s to form mono- and di-hydroxyflavones and that direct oxidation occurs in these MeFs to form mono-hydroxylated products, oxidized at the A- or B-ring of MeF.

CYP52A23 from Candida albicans and its Substrate Preference for Fatty Acid Hydroxylation.

The pathogenic fungus Candida albicans contains genes encoding five fatty acid hydroxylases belonging to the CYP52 family in its genome. Our previous study reported that CYP52A21 demonstrated typical omega-hydroxylation of lauric acid (Kim D, Cryle MJ, De Voss JJ, Ortiz de Montellano PR (2007) Arch Biochem Biophys 464, 213-220). Functional characterization of CYP52 fatty acid hydroxylases was studied, and their selectivity for hydroxylation was analyzed. Genes for four other CYP52 members (CYP52A22, CYP52A23, CYP52A24, and CYP52C3) from C. albicans were cloned, and their recombinant enzymes were expressed in Escherichia coli. CO-binding spectral analyses showed that the functional P450 holoenzyme was obtained only in CYP52A23, while no holoenzyme peak was observed in the other three CYP52 enzymes. Spectral change of the type II binding was observed in purified CYP52A23 when titrated with fatty acids but none was observed with alkanes. The gas chromatography-mass spectrometry (GC-MS) analysis revealed that CYP52A23 predominantly exhibited omega-hydroxylation activity during the oxidation reaction of fatty acids. Interestingly, it was found that CYP52A23 preferred longer-chain fatty acids (stearic acid and arachidic acid) for its catalytic activities while CYP52A21 preferred mid-chain fatty acids (lauric acid and mystic acid). To analyze the selectivity of fatty acids, hybrid mutagenesis of genes encoding CYP52A21 and CYP52A23 by overlap extension polymerase chain reaction was conducted. Two hybrid mutants containing the N-terminal fragments of CYP52A21 and C-terminal fragments of CYP52A23 displayed higher catalytic activity in palmitic acid and arachidic acid. These results suggested that the C-terminal part of CYP52A23 may be responsible for its preference to longer-chain fatty acids.

Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.

1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min-1 and 1.3 min-1, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min-1 that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min-1). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes.

The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2'-hydroxy-, 4'-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2'-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2'-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2' and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2, *3, *4, and *10.

Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower kcat values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased Km values and diminished kcat values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.

Structural Insights into the Interaction of Terpenoids with Streptomyces avermitilis CYP107P2.

Streptomyces avermitilis genome includes 33 genes encoding monooxygenation-catalyzing cytochrome P450 enzymes. We investigated the structure of CYP107P2 and its interactions with terpenoid compounds. The recombinant CYP107P2 protein was expressed in Escherichia coli and the purified enzyme exhibited a typical P450 spectrum upon CO-binding in its reduced state. Type-I substrate-binding spectral titrations were observed with various terpenoid compounds, including α-pinene, ß-pinene, α-terpinyl acetate, and (+)-3-carene. The calculated binding affinities (Kd) ranged from 15.9 to 50.8 µM. The X-ray crystal structure of CYP107P2 was determined at 1.99 Å resolution, with a well-conserved overall P450 folding conformation. The terpenoid compound docking models illustrated that the structural interaction between monoterpenes and CYP107P2, with the distance between heme and terpenes ranging from 3.4 to 5.4 Å, indicates potential substrate binding for P450 enzyme. This study suggests that CYP107P2 is a Streptomyces P450 enzyme capable of catalyzing terpenes as substrates, signifying noteworthy advancements in comprehending a novel P450 enzyme's involvement in terpene reactions.

Catalytic enhancements in cytochrome P450 2C19 by cytochrome b5.

Human cytochrome P450 2C19 catalyzes P450 enzyme reactions of various substrates, including steroids and clinical drugs. Recombinant P450 2C19 enzyme with histidine tag was successfully expressed in Escherichia coli and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mass (UPLC-MS/MS) spectrometry showed that the purified P450 2C19 enzyme catalyzed 5-hydroxylation reaction of omeprazole. The purified enzyme displayed typical type I binding spectra to progesterone with a Kd value of 4.5 ± 0.2 µM, indicating a tight substrate binding. P450 2C19 catalyzed the hydroxylation of progesterone to produce 21-hydroxy (OH) as a major and 17-OH product as a minor product. Steady-state kinetic analysis of progesterone 21-hydroxylation indicated that the addition of cytochrome b5 stimulated a five-times catalytic turnover number of P450 2C19 with a kcat value of 1.07 ± 0.08 min-1. The molecular docking model of progesterone in the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme with a distance of 4.7 Å, suggesting 21-hydroxylation of progesterone is the optimal reaction of P450 2C19 enzyme for a productive orientation of the substrate. Our findings will help investigate the extent to which cytochrome b5 affects the metabolism of P450 2C19 to drugs and steroids. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00219-8.

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1).

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.

Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase.

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni2+-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with Kd values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a kcat value of 0.038 ± 0.002 min-1 and a Km value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a kcat value of 0.135 ± 0.007 min-1 and a Km value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

Structure-Functional Analysis of Human Cytochrome P450 2C8 Using Directed Evolution.

The human genome includes four cytochrome P450 2C subfamily enzymes, and CYP2C8 has generated research interest because it is subject to drug-drug interactions and various polymorphic outcomes. To address the structure-functional complexity of CYP2C8, its catalytic activity was studied using a directed evolution analysis. Consecutive rounds of random mutagenesis and screening using 6-methoxy-luciferin produced two mutants, which displayed highly increased luciferase activity. Wild-type and selected mutants were expressed on a large scale and purified. The expression levels of the D349Y and D349Y/V237A mutants were ~310 and 460 nmol per liter of culture, respectively. The steady-state kinetic analysis of paclitaxel 6α-hydroxylation showed that the mutants exhibited a 5-7-fold increase in kcat values and a 3-5-fold increase in catalytic efficiencies (kcat/KM). In arachidonic acid epoxidation, two mutants exhibited a 30-150-fold increase in kcat values and a 40-110-fold increase in catalytic efficiencies. The binding titration analyses of paclitaxel and arachidonic acid showed that the V237A mutation had a lower Kd value, indicating a tighter substrate-binding affinity. The structural analysis of CYP2C8 indicated that the D349Y mutation was close enough to the putative binding domain of the redox partner; the increase in catalytic activity could be partially attributed to the enhancement of the P450 coupling efficiency or electron transfer.