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.

Oxidation of Flavone, 5-Hydroxyflavone, and 5,7-Dihydroxyflavone to Mono-, Di-, and Tri-Hydroxyflavones by Human Cytochrome P450 Enzymes.

Biologically active plant flavonoids, including 5,7-dihydroxyflavone (57diOHF, chrysin), 4',5,7-trihydroxyflavone (4'57triOHF, apigenin), and 5,6,7-trihydroxyflavone (567triOHF, baicalein), have important pharmacological and toxicological significance, e.g., antiallergic, anti-inflammatory, antioxidative, antimicrobial, and antitumorgenic properties. In order to better understand the metabolism of these flavonoids in humans, we examined the oxidation of flavone, 5-hydroxyflavone (5OHF), and 57diOHF to various products by human cytochrome P450 (P450 or CYP) and liver microsomal enzymes. Individual human P450s and liver microsomes oxidized flavone to 6-hydroxyflavone, small amounts of 5OHF, and 11 other monohydroxylated products at different rates and also produced several dihydroxylated products (including 57diOHF and 7,8-dihydroxyflavone) from flavone. We also found that 5OHF was oxidized by several P450 enzymes and human liver microsomes to 57diOHF and further to 567triOHF, but the turnover rates in these reactions were low. Interestingly, both CYP1B1.1 and 1B1.3 converted 57diOHF to 567triOHF at turnover rates (on the basis of P450 contents) of >3.0 min-1, and CYP1A1 and 1A2 produced 567triOHF at rates of 0.51 and 0.72 min-1, respectively. CYP2A13 and 2A6 catalyzed the oxidation of 57diOHF to 4'57triOHF at rates of 0.7 and 0.1 min-1, respectively. Our present results show that different P450s have individual roles in oxidizing these phytochemical flavonoids and that these reactions may cause changes in their biological and toxicological properties in mammals.

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.

Oxidation of 1-chloropyrene by human CYP1 family and CYP2A subfamily cytochrome P450 enzymes: catalytic roles of two CYP1B1 and five CYP2A13 allelic variants.

1. 1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2. CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3. Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower kcat value than other CYP2A13.1 variants. 4. CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5. Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2 A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.

Structural insights into the binding of lauric acid to CYP107L2 from Streptomyces avermitilis.

Streptomyces avermitilis is an actinobacterium known to produce clinically useful macrolides including avermectins. CYP107L2 from S. avermitilis shares a high sequence similarity with the PikC (CYP107L1) from S. venezuelae. To elucidate the structural features of CYP107L2, we conducted biochemical and structural characterization of CYP107L2 from S. avermitilis. The CYP107L2 gene was cloned, and its recombinant protein was expressed and purified. The CYP107L2 showed a low-spin state of heme, and the reduced form yielded the CO difference spectra with a maximal absorption at 449 nm. Binding of pikromycin and lauric acid yielded the typical type I spectra with Kd values of 4.8 ± 0.3 and 111 ± 9 µM, respectively. However, no metabolic product was observed in the enzyme reaction. X-ray crystal structures of the ligand-free CYP107L2 and its complex with lauric acid were determined at the resolution of 2.6 and 2.5 Å, respectively. CYP107L2 showed a well-conserved CYP structure with a wide-open substrate-binding cavity. The lauric acid is bound mainly via hydrophobic interactions with the carboxylate group of lauric acid coordinated to the heme of P450. Glu-40 and Leu-382 residues in the CYP107L2 complex with lauric acid showed significant conformational changes to provide plentiful room for the lauric acid in the substrate-binding site.

Structure-Function Studies of Naphthalene, Phenanthrene, Biphenyl, and Their Derivatives in Interaction with and Oxidation by Cytochromes P450 2A13 and 2A6.

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.

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.

Directed-evolution analysis of human cytochrome P450 2A6 for enhanced enzymatic catalysis.

Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation. Four consecutive rounds of random mutagenesis and screening were performed and catalytically enhanced mutants were selected in each round of screening. The selected mutants showed the sequentially accumulated mutations of amino acid residues of P450 2A6: B1 (F209S), C1 (F209S, S369G), D1 (F209S, S369G, E277K), and E1 (F209S, S369G, E277K, A10V). E1 mutants displayed approximately 13-fold increased activity based on fluorescent coumarin hydroxylation assays at bacterial whole cell level. Steady-state kinetic parameters for coumarin 7-hydroxylation and nicotine oxidation were measured in purified mutant enzymes and indicated catalytic turnover numbers (kcat) of selected mutants were enhanced up to sevenfold greater than wild-type P450 2A6. However, all mutants displayed elevated Km values and therefore catalytic efficiencies (kcat/Km) were not improved. The increase in Km values was partially attributed to reduction in substrate binding affinities measured in the analysis of substrate binding titration. The structural analysis of P450 2A6 indicates that F209S mutation is sufficient to affect direct interaction of substrate at the active site.

Crystal structure of cytochrome P450 CYP105N1 from Streptomyces coelicolor, an oxidase in the coelibactin siderophore biosynthetic pathway.

The genome sequence of Streptomyces coelicolor contains 18 cytochrome P450 enzymes. The recombinant CYP105N1 protein has been expressed in Escherichia coli and purified, and we report the biochemical and structural characterization of CYP105N1 from S. coelicolor. The purified protein exhibited the typical CO-binding spectrum of P450 enzymes and type I binding spectra with estradiol and a coelibactin analog. The oxidation of estradiol by CYP105N1, supported by H(2)O(2), produced estriol. The crystal structure of CYP105N1 was determined at 2.9 Å resolution. An unexpected wide open binding pocket located above the heme group was identified, with a volume of approximately 4299 Å(3). These results suggest that the large open pocket to the active site may be a key feature for easy access of the peptidyl carrier protein-bound substrate to perform the hydroxylation reaction. A molecular docking model with coelibactin showed that the phenyl group of coelibactin is located <4 Å away from the heme-iron, suggesting that CYP105N1 may be involved in the hydroxylation of the phenyl ring of the coelibactin precursor during biosynthesis.

Functional expression and characterization of CYP51 from dandruff-causing Malassezia globosa.

Malassezia globosa is one of the most common yeasts to cause various human skin diseases including dandruff and seborrheic dermatitis. Genomic analysis of M. globosa revealed four putative cytochrome P450 (CYP) enzymes. Here, we report the purification and characterization of recombinant CYP51, a putative lanosterol 14α-demethylase, from M. globosa. The M. globosa CYP51 was expressed heterologously in Escherichia coli, followed by purification. Purified CYP51 showed a typical reduced CO-difference spectrum of P450, with a maximum absorption at 447 nm. Purified CYP51 exhibited tight binding to azole antifungal agents such as ketoconazole, econazole, fluconazole, or itraconazole, with K(d) values around 0.26-0.84 µM, which suggests that CYP51 is an orthologous target for antifungal agents in the M. globosa. In addition, three mutations (Y127F, A169S, and K176N) in the amino acid sequence of M. globosa CYP51 were identified in one of the azole-resistant strains. Homology modeling of M. globosa CYP51 suggested that the Y127F mutation may influence the resistance to azoles by blocking substrate access channels. Taken together, functional expression and characterization of the CYP51 enzyme can provide a fundamental basis for a specific antifungal drug design for dandruff caused by M. globosa.

Candida albicans NADPH-P450 reductase: expression, purification, and characterization of recombinant protein.

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6x(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.

Regioselective oxidation of lauric acid by CYP119, an orphan cytochrome P450 from Sulfolobus acidocaldarius.

Archaebacteria Sulfolobus acidocaldarius contains the highly thermophilic cytochrome P450 enzyme (CYP119). CYP119 possesses stable enzymatic activity at up to 85 degrees C. However, this enzyme is still considered as an orphan P450 without known physiological function with endogenous or xenobiotic substrates. We characterized the regioselectivity of lauric acid by CYP119 using the auxiliary redox partner proteins putidaredoxin (Pd) and putidaredoxin reductase (PdR). Purified CYP119 protein showed a tight binding affinity to lauric acid (K(d)=1.1+/-0.1 microM) and dominantly hydroxylated (omega-1) position of lauric acid. We determined the steady-state kinetic parameters; k(cat) was 10.8 min(-1) and K(m) was 12 microM. The increased ratio to omega-hydroxylated production of lauric acid catalyzed by CYP119 was observed with increase in the reaction temperature. These studies suggested that the regioselectivity of CYP119 provide the critical clue for the physiological enzyme function in this thermophilic archaebacteria. In addition, regioselectivity control of CYP119 without altering its thermostability can lead to the development of novel CYP119-based catalysts through protein engineering.

Streptomyces Cytochrome P450 Enzymes and Their Roles in the Biosynthesis of Macrolide Therapeutic Agents.

The study of the genus Streptomyces is of particular interest because it produces a wide array of clinically important bioactive molecules. The genomic sequencing of many Streptomyces species has revealed unusually large numbers of cytochrome P450 genes, which are involved in the biosynthesis of secondary metabolites. Many macrolide biosynthetic pathways are catalyzed by a series of enzymes in gene clusters including polyketide and non-ribosomal peptide synthesis. In general, Streptomyces P450 enzymes accelerate the final, post-polyketide synthesis steps to enhance the structural architecture of macrolide chemistry. In this review, we discuss the major Streptomyces P450 enzymes research focused on the biosynthetic processing of macrolide therapeutic agents, with an emphasis on their biochemical mechanisms and structural insights.

Terfenadine metabolism of human cytochrome P450 2J2 containing genetic variations (G312R, P351L and P115L).

The human cytochrome P450 2J2 is involved in several metabolic reactions, including the oxidation of important therapeutics and epoxidation of endogenous arachidonic acid. At least ten genetic variations of P450 2J2 have been identified, but their effects on enzymatic activity have not been clearly characterized. Here, we evaluated the functional effects of three genetic variations of P450 2J2 (G312R, P351L, and P115L). Recombinant enzymes of wild-type and three variant P450 2J2 were heterologously expressed in Escherichia coli and purified. P450 expression levels in the wild-type and two variants (P351L and P115L) were 142-231 nmol per liter culture, while the G312R variant showed no holoenzyme peak in the CO-binding spectra. Substrate binding titrations to terfenadine showed that the wild-type and two variants displayed Kd values of 0.90-2.2 µM, indicating tight substrate binding affinities. Steady-state kinetic analysis for t-butyl methyl hydroxylation of terfenadine indicated that two variant enzymes had similar kcat and Km values to wild-type P450 2J2. The locations of mutations in three-dimensional structural models indicated that the G312R is located in the I-helix region near the formal active site in P450 2J2 and its amino acid change affected the structural stability of the P450 heme environment.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP+ in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni2+-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Terpenes from Forests and Human Health.

Forest bathing has beneficial effects on human health via showering of forest aerosols as well as physical relaxation. Terpenes that consist of multiple isoprene units are the largest class of organic compounds produced by various plants, and one of the major components of forest aerosols. Traditionally, terpene-containing plant oil has been used to treat various diseases without knowing the exact functions or the mechanisms of action of the individual bioactive compounds. This review categorizes various terpenes easily obtained from forests according to their anti-inflammatory, anti-tumorigenic, or neuroprotective activities. Moreover, potential action mechanisms of the individual terpenes and their effects on such processes, which are described in various in vivo and in vitro systems, are discussed. In conclusion, the studies that show the biological effectiveness of terpenes support the benefits of forest bathing and propose a potential use of terpenes as chemotherapeutic agents for treating various human diseases.

The N-terminal region of organic anion transporting polypeptide 1B3 (OATP1B3) plays an essential role in regulating its plasma membrane trafficking.

Organic anion transporting polypeptide 1B3 (OATP1B3) is a major influx transporter mediating the hepatic uptake of various endogenous substrates as well as clinically important drugs such as statins and anticancer drugs. However, molecular mechanisms controlling the membrane trafficking of OATP1B3 have been largely unknown. Several reports recently indicated the presence of a distinct, cancer-type OATP1B3 variant lacking the N-terminal 28 amino acids compared to OATP1B3 expressed in non-malignant hepatocytes. Interestingly, the cancer-type OATP1B3 variant is located predominantly in the cytoplasm, implicating the involvement of the N-terminal region of OATP1B3 in its membrane trafficking. In the current study, we set out to experimentally validate the importance of the N-terminal region of OATP1B3 and to identify responsible sequence motif(s) in that region. A number of truncation or point mutants of OATP1B3 were transiently expressed in HEK293T, HCT-8 or MDCK II cells and their expression in cytoplasmic and surface membrane fractions were analyzed by immunoblotting. Our results indicated that the N-terminal sequence of OATP1B3, in particular, at the amino acid positions between 12 and 28, may be indispensable in its membrane trafficking. Moreover, our results using a fusion construct indicated that the first 50 amino acids of OATP1B3 are sufficient for its membrane localization. The importance of the N-terminal region in membranous localization was shared among the other OATP1B subfamily members, OATP1B1 and rat Oatp1b2. Our efforts to identify the responsible amino acid(s) or structure motif(s) in the N-terminal region did not pinpoint individual amino acids or motifs with putative secondary structures. Our current findings however demonstrate that the N-terminal region is important for the membrane localization of the OATP1B subfamily members and should facilitate future investigations of the mechanisms involved in the regulation and membrane trafficking of these important transporter proteins.

Characterization of a Biflaviolin Synthase CYP158A3 from Streptomyces avermitilis and Its Role in the Biosynthesis of Secondary Metabolites.

Streptomyces avermitilis produces clinically useful drugs such as avermectins and oligomycins. Its genome contains approximately 33 cytochrome P450 genes and they seem to play important roles in the biosynthesis of many secondary metabolites. The SAV_7130 gene from S. avermitilis encodes CYP158A3. The amino acid sequence of this enzyme has high similarity with that of CYP158A2, a biflaviolin synthase from S. coelicolor A3(2). Recombinant S. avermitilis CYP158A3 was heterologously expressed and purified. It exhibited the typical P450 Soret peak at 447 nm in the reduced CO-bound form. Type I binding spectral changes were observed when CYP158A3 was titrated with myristic acid; however, no oxidative product was formed. An analog of flaviolin, 2-hydroxynaphthoquinone (2-OH NQ) displayed similar type I binding upon titration with purified CYP158A3. It underwent an enzymatic reaction forming dimerized product. A homology model of CYP158A3 was superimposed with the structure of CYP158A2, and the majority of structural elements aligned. These results suggest that CYP158A3 might be an orthologue of biflaviolin synthase, catalyzing C-C coupling reactions during pigment biosynthesis in S. avermitilis.

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.