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.

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.

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.

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.

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.

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.

Functional expression in Bacillus subtilis of mammalian NADPH-cytochrome P450 oxidoreductase and its spore-display.

The technology for over-expressing NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, offers the opportunity to develop enzymatic systems for environmental detoxication and bioconversions of drugs, pesticides and fine chemicals. In this study, Bacillus subtilis was chosen to express rat CPR (rCPR) because of its capacities for high protein production and spore formation. rCPR was expressed in B. subtilis DB104 under the transcriptional control of an IPTG-inducible fusion promoter of P(groE) and P(tac). The expressed rCPR was released into the culture medium after sporulation by autolysis of the host cell. It was associated with and displayed on the spore surfaces; this was confirmed by measuring rCPR activity in purified spores and analyzing its accessibility to anti-rCPR antibodies using flow cytometry. The spore-displayed rCPR was able to reduce cytochrome c and ferricyanide, and also assisted in the O-deethylation of 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin (EFC) by human cytochrome P450 1A2, indicating that it was functionally active. Spore surface display of rCPR in B. subtilis appears to be useful for preparing cytochrome P450-related enzymes, and spore biocatalysts of rCPR are likely to have wide biotechnological applications.

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.

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.

Entrapment of cytochrome P450 BM-3 in polypyrrole for electrochemically-driven biocatalysis.

In vitro biocatalysis with cytochrome P450 BM-3 was investigated aiming for the substitution of the expensive natural cofactor NADPH by electrochemistry. The monooxygenase was immobilized on electrodes by entrapment in polypyrrole as a conductive polymer for electrochemically wiring the enzyme. Electropolymerization of pyrrole proved to be a useful means of immobilising an active cytochrome P450 BM-3 mutein on platinum and glassy carbon electrodes without denaturation. Repeatedly sweeping the electric potential between -600 and +600 mV versus Ag/AgCl led to enzymatically-catalysed product formation while in the absence of the enzyme no product formed under otherwise identical conditions.

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.

NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability.

NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.

Bioreduction of idarubicin and formation of ROS responsible for DNA cleavage by NADPH-cytochrome P450 reductase and its potential role in the antitumor effect.

PURPOSE: Idarubicin is a clinically effective synthetic anthracycline analog used in the treatment of several human neoplasms. Anthracyclines have the potential to undergo bioactivation by flavoenzymes to free radicals and thus exert their cytotoxic actions. In this study, our main objective was to investigate the possible involvement of NADPH-cytochrome P450 reductase in the bioreductive activation of idarubicin to DNA-damaging species. METHODS: A pBR322 plasmid DNA damage assay was used as a sensitive method for detecting strand breaks in DNA exposed to idarubicin in the presence of P450 reductase and cofactor NADPH under various incubation conditions. In addition, the rates of idarubicin reduction by P450 reductases purified from phenobarbital-treated rabbit liver, beef liver and sheep lung microsomes were determined by measuring NADPH oxidation at 340 nm. RESULTS: The plasmid DNA experiments demonstrated that idarubicin could undergo bioreduction by P450 reductase with the resulting formation of DNA strand breaks. The antioxidant enzymes SOD and catalase, and hydroxyl radical scavengers, DMSO and thiourea, afforded significant levels of protection against idarubicin-induced DNA strand breaks. These findings suggested that DNA damage by idarubicin occurs through a mechanism which involves its redox cycling with P450 reductase to generate reactive oxygen species (ROS). The extent of DNA damage by idarubicin was found to increase with increasing concentrations of drug or enzyme as well as with increasing incubation time. The capacity of idarubicin to induce DNA damage under above incubation conditions was compared with that of a model compound, mitomycin C. Finally, enzyme assays carried out with purified P450 reductases revealed that idarubicin exhibited about two-fold higher rate of reduction than mitomycin C. CONCLUSION: Our findings implicated bioreduction of idarubicin by P450 reductase and subsequent redox cycling under aerobic conditions as being one mode of idarubicin action potentially contributing to its antitumor effect.

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.

Purification of NADPH-P450 reductase (NPR) from Xenopus laevis and the developmental change in NPR expression.

NADPH-P450 reductase (NPR) was purified from hepatic microsomes of Xenopus laevis. The electron transfer activity of purified NPR was 23.8 units/min/mg with horse cytochrome c. The aminopyrine demethylation activity of rat CYP2B1 with Xenopus NPR was 58.1 nmol/min/nmol. The corresponding cDNA was isolated from Xenopus liver. The homology in amino acid sequence deduced from NPR cDNA isolated from Xenopus liver was 80%, 78%, and 81% with human, rat, and rabbit NPR, respectively. Antibody against Xenopus NPR was prepared. The expression of NPR was investigated in various tissues and in early development by Western blotting. NPR was most abundantly expressed in the kidney, followed by the liver, lung, and heart. The brain had very low levels of NPR. The level of NPR protein was almost the same at all stages, 2-cell stage (st. 2), blastula (st. 8), gastrula (st. 12), tail bud (st. 26) and larva (st.35), examined in this study. We further investigated the distribution of NPR using whole-mount in situ hybridization. NPR mRNA was expressed in cement gland, lens placode, ear vesicle, mesencephalon, rhombencephalon, lymphatic vessel, and heart anlage in the embryo at stage 29. Xenopus NPR has similar properties to mouse and rat NPRs. Localization of NPR in Xenopus embryo was consistent with the abnormal region caused by NPR deficiency in mice.

Cytochromes P450 reconstituted with NADPH: P450 reductase mimic the activating and detoxicating metabolism of the anticancer drug ellipticine in microsomes.

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple action mechanisms. Recently, we found that after cytochrome P450 (CYP)-mediated oxidation ellipticine forms covalent DNA adducts. Ellipticine oxidation by isolated CYP and its binding to DNA is the target of this study. METHODS: High performance liquid chromatography (HPLC) was employed for separation and characterization of ellipticine metabolites generated by CYPs. The (32)P-postlabeling technique was utilized to determine ellipticine-DNA adducts. RESULTS: Purified CYP enzymes reconstituted with NADPH:CYP reductase oxidized ellipticine to up to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only CYP1A1 was capable to form all metabolites. Using the reconstituted enzymatic system, we demonstrated that the detoxication ellipticine metabolites, 7-hydroxyellipticine and 9-hydroxyellipticine, are mainly generated by CYP1A1 and 1A2, while those responsible for DNA binding, 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, by CYP3A1 and 2C3. Likewise, the most efficient CYPs forming DNA adducts from ellipticine were CYP3A1 and 2C3. CONCLUSIONS: The results showed that the system of purified CYPs reconstituted with NADPH: CYP reductase proved for ellipticine oxidation provide a true reflection of the situation in the microsomal membrane.