Characterization of Two Self-Sufficient Monooxygenases, CYP102A15 and CYP102A170, as Long-Chain Fatty Acid Hydroxylases.

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named CYP102A15 and CYP102A170, from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724, respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C12-C20, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with Km 15.3 µM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 µM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.

Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum.

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5-9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5-8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

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.

Single-Protein Tracking Reveals That NADPH Mediates the Insertion of Cytochrome P450 Reductase into a Biomimetic of the Endoplasmic Reticulum.

Cytochrome P450 reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein, we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid-supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature-dependent fluorescence recovery after photobleaching. The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single-molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramatic changes in diffusion coefficient and the degree of partitioning of CPR as a function of NADPH concentration.

Quantification of Functional Dynamics of Membrane Proteins Reconstituted in Nanodiscs Membranes by Single Turnover Functional Readout.

Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge, and to date there is limited number of available functional assays. In this chapter, we describe in detail our recently developed methodology to reconstitute membrane proteins such as the integral membrane protein cytochrome P450 oxidoreductase on membrane systems such as Nanodiscs and study their functional dynamics by recordings at the fundamental resolution of individual catalytic turnovers using prefluorescent substrate analogues. We initially describe the methodology for reconstitution, surface immobilization, and data acquisition of individual enzyme catalytic turnovers. We then explain in detail the statistical analysis, with an emphasis on the model development, the potential pitfalls for correctly identifying the abundance, lifetime, and likelihood of sampling protein functional states. This methodology may enable studies of functional dynamics and their role in biology for a spectrum of membrane proteins.

Membrane anchor of cytochrome P450 reductase suppresses the uncoupling of cytochrome P450.

Cytochrome P450 reductase (CPR) is an important redox partner of microsomal CYPs. CPR is composed of a membrane anchor and a catalytic domain that contains FAD and flavin mononucleotide (FMN) as redox centers and mediates electron transfer to CYP. Although the CPR membrane anchor is believed to be requisite for interaction with CYP, its physiological role is still controversial. To clarify the role of the anchor, we constructed a mutant (Δ60-CPR) in which the N-terminal membrane anchor was truncated, and studied its effect on binding properties, electron transfer to CYP2C19, and drug metabolism. We found that Δ60-CPR could bind to and transfer electrons to CYP2C19 as efficiently as WT-CPR, even in the absence of lipid membrane. In accordance with this, Δ60-CPR could mediate metabolism of amitriptyline (AMT) and imipramine (IMP) in the absence of lipids, although activity was diminished. However, Δ60-CPR failed to metabolize omeprazole (OPZ) and lansoprazole (LPZ). To clarify the reason for this discrepancy in drug metabolism, we investigated the uncoupling reaction of the CYP catalytic cycle. By measuring the amount of H2O2 by-product, we found that shunt pathways were markedly activated in the presence of OPZ/LPZ in the Δ60-CPR mutant. Because H2O2 levels varied among the drugs, we conclude that the proton network in the distal pocket of CYP2C19 is perturbed differently by different drugs, and activated oxygen is degraded to become H2O2. Therefore, we propose a novel role for the membrane anchor as a suppressor of the uncoupling reaction in drug metabolism by CYP.

Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis.

We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a monohydroxylated product is detected by gas chromatography. Hydroxylation occurs exclusively at the allylic position as confirmed from a synthesized authentic standard. Investigation into the stereochemistry of the reaction indicates that the R enantiomer is obtained in 85% ee. The (R)-9-hydroxy-10-undecenoic acid obtained enzymatically is a valuable synthon en route to various natural products further expanding the light-activated P450 BM3 biocatalysis and highlighting the advantages over traditional methods.

P450 oxidoreductase: genotyping, expression, purification of recombinant protein, and activity assessments of wild-type and mutant protein.

P450 oxidoreductase (POR) is the flavoprotein that transfers electrons from NADPH to microsomal cytochrome P450 enzymes and to some other proteins. Protocols for genotyping human POR for common polymorphisms are described. Expression in E. coli of recombinant human POR, its purification, and different methods of assessing the effect of amino-acid sequence variants of POR on the activity of various cytochromes P450 are also described.

Hydroxylation of non-substituted polycyclic aromatic hydrocarbons by cytochrome P450 BM3 engineered by directed evolution.

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene. The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach. The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.

Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

High-yield expression of a catalytically active membrane-bound protein: human P450 oxidoreductase.

P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 µm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 µm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins.

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca.

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.

Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4.

We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 µM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s(-1). Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6ß-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 µM. This suggests that the CPR-CYP3A4 complex can dissociate and reform between the first and second electron transfers.

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.

P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins.

With an aim to understand the cytochrome P450 enzyme system in the white rot fungus Phanerochaete chrysosporium, here we report molecular characterization of its P450 redox proteins including the primary P450 oxidoreductase (POR) and two alternate P450 redox proteins cytochrome b5 (cyt b5) and cytochrome b5 reductase (cyt b5r) in terms of transcriptional regulation and heterologous expression. The transcript abundance followed the order POR > cyt b5r > cyt b5. Interestingly, the three genes showed an overall higher expression in the defined carbon-limited cultures with low nitrogen (LN) or high nitrogen (HN) versus the carbon-rich malt extract (ME) cultures. cDNA cloning and analysis revealed the following deduced protein characteristics: cyt b5 (238 amino acids, 25.38 kDa) and cyt b5r (321 amino acids, 35.52 kDa). Phylogenetic analysis revealed that the cloned cyt b5 belongs to a novel class of fungal cyt b5-like proteins. The two proteins cyt b5 and cyt b5r were heterologously expressed in E. coli and purified using affinity-based purification in an active form. The POR was heterologously expressed in Saccharomyces cerevisiae and was also purified in active form as evidenced by its cytochrome c reduction activity. This is the first report on cloning, heterologous expression, and purification of the alternate redox proteins cyt b5 and cyt b5r in E. coli and on yeast expression of POR from this model white rot fungus.

[cDNA cloning, heterologous overexpression and activity analysis of cytochrome P450 reductase of Taxus chinensis].

NADPH-cytochrome P450 reductase (CPR), a partner for P450 monooxygenases, serves as the electron donor to almost all eukaryotic cytochrome P450s. One cDNA (TchCPR) encoding cytochrome P450 reductase of T. chinensis was isolated from callus cells. The cDNA contains an open reading frame of 2154 nucleotides which encodes a protein of 717 amino acid residues. The TchCPR has higher similarity to other CPRs of gumnosperms (>82%) than that of angiosperms (<74%). The recombinant full-length TchCPR and a series of N-terminal truncated constructs with N-terminal fusion of His Tag were obtained and induced to express in E. coli B121(DE3), and then purified using affinity chromatography. The truncated forms of N-terminal more than 61 amino acid residues could be efficiently expressed while the truncated mutant of N-terminal 48 amino acid residues and the wild-type TchCPR were not successfully expressed in E. coli cells. The activity of the truncated TchCPR was assayed by measuring the reduction of cytochrome C. The electron transfer activity of the recombinantly purified CPRT61 was 1.6057 nmol of cytochrome C reduced per min per microg TchCPR reductase, and it is higher than that of the other four truncated forms.

Characterization of two NADPH: cytochrome P450 reductases from cotton (Gossypium hirsutum).

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.

Purification, cDNA cloning and functional expression of NADPH-cytochrome P450 reductase from Centaurium erythraea cell cultures.

Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury (Centaurium erythraea) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N-glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation.

Probing the molecular determinants of coenzyme selectivity in the P450 BM3 FAD/NADPH domain.

Bacillus megaterium P450 BM3 (BM3) is an NAD(P)H-binding diflavin reductase exhibiting substantial coenzyme specificity for NADPH over NADH. The side chains of serine 965, arginine 966 and lysine 972 in its FAD-binding domain bind the NADPH 2'-phosphate. Optical, kinetic and thermodynamic properties of S965A, R966A and K972A FAD domains were analyzed singly and combined with the FAD-shielding W1046A mutation. Steady-state and stopped-flow kinetic studies demonstrated substantially decreased NADPH affinity versus wild-type (WT) FAD domain (146-fold for the S965A K(d)). Considerable catalytic efficiency increases (the ratio of specificity constants, k(cat)/K(m), for the coenzymes) with NADH were observed for each point mutant over WT (570-fold in K972A), along with increased rates of NADH-dependent FAD reduction (k(lim) elevated 5.2-fold in R966A). In combination with W1046A, considerable (37 to 56-fold) improvements over WT were seen in the k(lim) parameters with NADH for all double mutants. Each 2'-phosphate binding point mutant produced large increases in FAD potential (111 mV in R966A), despite large distances between these residues and the FAD isoalloxazine ring (18-21 A), suggesting long range conformational influences on FAD environment. The W1046A/K972A mutant abolished NADPH selectivity (8340-fold coenzyme selectivity switch towards NADH), with ramifications for BM3's biotechnological exploitation using the cheaper NADH coenzyme.

Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures.