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The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.

International audience; Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and β-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1−/− mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an “orphan” P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can "pull" substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O2 activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.

Since the discovery of Acanthamoeba polyphaga Mimivirus, the first giant virus of amoeba, the historical hallmarks defining a virus have been challenged. Giant virion sizes can reach up to 2.3 µm, making them visible by optical microscopy. They have large genomes of up to 2.5 Mb that encode proteins involved in the translation apparatus. Herein, we investigated possible energy production in Pandoravirus massiliensis, the largest of our giant virus collection. MitoTracker and TMRM mitochondrial membrane markers allowed for the detection of a membrane potential in virions that could be abolished by the use of the depolarizing agent CCCP. An attempt to identify enzymes involved in energy metabolism revealed that 8 predicted proteins of P. massiliensis exhibited low sequence identities with defined proteins involved in the universal tricarboxylic acid cycle (acetyl Co-A synthase; citrate synthase; aconitase; isocitrate dehydrogenase; α-ketoglutarate decarboxylase; succinate dehydrogenase; fumarase). All 8 viral predicted ORFs were transcribed together during viral replication, mainly at the end of the replication cycle. Two of these proteins were detected in mature viral particles by proteomics. The product of the ORF132, a predicted protein of P. massiliensis, cloned and expressed in Escherichia coli, provided a functional isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle, which converts isocitrate to α-ketoglutarate. We observed that membrane potential was enhanced by low concentrations of Acetyl-CoA, a regulator of the tricarboxylic acid cycle. Our findings show for the first time that energy production can occur in viruses, namely, pandoraviruses, and the involved enzymes are related to tricarboxylic acid cycle enzymes. The presence of a proton gradient in P. massiliensis coupled with the observation of genes of the tricarboxylic acid cycle make this virus a form a life for which it is legitimate to question ‘what is a virus?’.

Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone".

Genes encoding cytochrome P450 (CYP; P450) enzymes occur widely in the Archaea, Bacteria, and Eukarya, where they play important roles in metabolism of endogenous regulatory molecules and exogenous chemicals. We now report that genes for multiple and unique P450s occur commonly in giant viruses in the Mimiviridae , Pandoraviridae , and other families in the proposed order Megavirales. P450 genes were also identified in a herpesvirus ( Ranid herpesvirus 3 ) and a phage ( Mycobacterium phage Adler). The Adler phage P450 was classified as CYP102L1, and the crystal structure of the open form was solved at 2.5 Å. Genes encoding known redox partners for P450s (cytochrome P450 reductase, ferredoxin and ferredoxin reductase, and flavodoxin and flavodoxin reductase) were not found in any viral genome so far described, implying that host redox partners may drive viral P450 activities. Giant virus P450 proteins share no more than 25% identity with the P450 gene products we identified in Acanthamoeba castellanii , an amoeba host for many giant viruses. Thus, the origin of the unique P450 genes in giant viruses remains unknown. If giant virus P450 genes were acquired from a host, we suggest it could have been from an as yet unknown and possibly ancient host. These studies expand the horizon in the evolution and diversity of the enormously important P450 superfamily. Determining the origin and function of P450s in giant viruses may help to discern the origin of the giant viruses themselves.

Ultra-thin CdTe photovoltaic solar cells with an absorber thickness of 0.5 μm were produced by metal organic chemical vapour deposition onto indium tin oxide coated boroaluminosilicate glass. A wide band gap Cd1−xZnxS alloy window layer was employed to improve spectral response in the blue region of the solar spectrum. X-ray photoelectron spectroscopy, X-ray diffraction and scanning electron microscopy were used to monitor changes in the chemical composition and microstructure of the Cd1−xZnxS/CdTe solar cell after varying the post-deposition CdCl2 activation treatment time and annealing temperature. The CdCl2 treatment leached Zn from the Cd1−xZnxS layer causing a redshift in the spectral response onset of window absorption. S diffusion occurred across the Cd1−xZnxS/CdTe interface, which was more pronounced as the CdCl2 treatment was increased. A CdTe1−ySy alloy was formed at the interface, which thickened with CdCl2 treatment time. Small concentrations of S (up to 2 at.%) were observed throughout the CdTe layer as the degree of CdCl2 treatment was increased. Greater S diffusion across the Cd1−xZnxS/CdTe interface caused the device open-circuit voltage (Voc) to increase. The higher Voc is attributed to enhanced strain relaxation and associated reduction of defects in the interface region as well as the increase in CdTe grain size.

Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.

The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed ‘bld’ (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules.

Biosynthesis of steroid hormones in vertebrates involves three cytochrome P450 hydroxylases, CYP11A1, CYP17A1 and CYP19A1, which catalyze sequential steps in steroidogenesis. These enzymes are conserved in the vertebrates, but their origin and existence in other chordate subphyla (Tunicata and Cephalochordata) have not been clearly established. In this study, selected protein sequences of CYP11A1, CYP17A1 and CYP19A1 were compiled and analyzed using multiple sequence alignment and phylogenetic analysis. Our analyses show that cephalochordates have sequences orthologous to vertebrate CYP11A1, CYP17A1 or CYP19A1, and that echinoderms and hemichordates possess CYP11-like but not CYP19 genes. While the cephalochordate sequences have low identity with the vertebrate sequences, reflecting evolutionary distance, the data show apparent origin of CYP11 prior to the evolution of CYP19 and possibly CYP17, thus indicating a sequential origin of these functionally related steroidogenic CYPs. Co-occurrence of the three CYPs in early chordates suggests that the three genes may have coevolved thereafter, and that functional conservation should be reflected in functionally important residues in the proteins. CYP19A1 has the largest number of conserved residues while CYP11A1 sequences are less conserved. Structural analyses of human CYP11A1, CYP17A1 and CYP19A1 show that critical substrate binding site residues are highly conserved in each enzyme family. The results emphasize that the steroidogenic pathways producing glucocorticoids and reproductive steroids are several hundred million years old and that the catalytic structural elements of the enzymes have been conserved over the same period of time. Analysis of these elements may help to identify when precursor functions linked to these enzymes first arose.

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered "orphan" CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to "deorphanization", that is, identifying CYP20A1 functions and its roles in health and disease.

Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the α-helical domain and the β-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation.

Albaflavenone, a tricyclic sesquiterpene antibiotic, is biosynthesized in Streptomyces coelicolor A3(2) by enzymes encoded in a two-gene operon. Initially, sesquiterpene cyclase catalyzes the cyclization of farnesyl diphosphate to the terpenoid epi-isozizaene, which is oxidized to the final albaflavenone by cytochrome P450 (CYP)170A1. Additionally, this CYP is a bifunctional enzyme, being able to also generate farnesene isomers from farnesyl diphosphate, owing to a terpene synthase active site moonlighting on the CYP molecule. To explore the functionality of this operon in other streptomycetes, we have examined culture extracts by GC/MS and established the presence of albaflavenone in five Streptomyces species. Bioinformatics examination of the predicted CYP170 primary amino acid sequences revealed substitutions in the CYP terpene synthase active site. To examine whether the terpene synthase site was catalytically active in another CYP170, we characterized the least related CYP170 orthologue from Streptomyces albus (CYP170B1). Following expression and purification, CYP170B1 showed a normal reduced CO difference spectrum at 450 nm, in contrast to the unusual 440-nm peak observed for S. coelicolor A3(2) CYP170A1. CYP170B1 can catalyze the conversion of epi-isozizaene to albaflavenone, but was unable to catalyze the conversion of farnesyl diphosphate to farnesene. Molecular modeling with our crystal structure of CYP170A1 suggests that the absence of key amino acids for binding the essential terpene synthase cofactor Mg(2+) may be the explanation for the loss of CYP170B1 bifunctionality.

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae , the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis . CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin “open” conformation of the enzyme ( K d [dissociation constant] of 0.1 μM), with binding to the low-spin “closed” form being significantly hindered ( K d of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly ( K d , 1.2 to 2.5 μM); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.

New 1-[(3-aryloxy-3-aryl)propyl]-1 H-imidazoles were synthesized and evaluated against Candida albicans and dermatophytes in order to develop structure-activity relationships (SARs). Against C. albicans the new imidazoles showed minimal inhibitory concentrations (MICs) comparable to those of ketoconazole, miconazole, and econazole, and were more potent than fluconazole. Several derivatives ( 10, 12, 14, 18- 20, 24, 28, 29, 30, and 34) turned out to be potent inhibitors of C. albicans strains resistant to fluconazole, with MIC values less than 10 microg/mL. Against dermatophytes strains, compounds 20, 25, and 33 (MICor= 5 microg/mL) were equipotent to ketoconazole, econazole, and miconazole. SARs of imidazoles 10- 44 were rationalized with reasonable accuracy by a previously developed quantitative pharmacophore for antifungal agents.

Cytochrome P450 158A2 (CYP158A2) has been shown to catalyze an unusual oxidative C-C coupling reaction to polymerize flaviolin and form highly conjugated pigments (three isomers of biflaviolin and one triflaviolin) in Streptomyces coelicolor A3(2) which protect the soil bacterium from deleterious effects of UV irradiation (Zhao B. et al. (2005) J. Biol. Chem. 280, 11599-11607). The present studies demonstrate that the subfamily partner CYP158A1, sharing 61% amino acid identity with CYP158A2, can also catalyze the same flaviolin dimerization reactions, but it generates just two of the three isomers of biflaviolin that CYP158A2 produces. Furthermore, the two CYP158A1 products have very different molar ratios compared with the corresponding CYP158A2 products, indicating that each enzyme maintains its own stereo- and regiospecificity. To find an explanation for these differences, three CYP158A1 structures have been solved by X-ray crystallography and have been compared with those for CYP158A2. The structures reveal surprising differences. Particularly, only one flaviolin molecule is present close to the heme iron in CYP158A1, and the second flaviolin molecule binds at the entrance of the putative substrate access channel on the protein distal surface 9 A away. Our work describes two members of the same P450 subfamily, which produce the same products by oxidative C-C coupling yet show very different structural orientations of substrate molecules in the active site.

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga . In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli . Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [ K d ] of 10 and 13 nM), whereas fluconazole bound weakly ( K d of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC 50 ] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC 50 , 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro . Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga ; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.

One of the surprising discoveries about the genomics of the cytochrome P450 (CYP) superfamily is the large number of CYPs in the bacterial class of actinomycetes. It had previously been imagined that bacteria have small numbers of CYPs or none at all. Particularly intriguing is that the bacterial genus Streptomyces, which produce a large number of secondary metabolites with important medical application, has a large CYP complement reflecting the ecological niche that the organism finds itself in. In 2001 the first complete Streptomyces species genome (Streptomyces coelicolor A3[2]) was published, revealing the presence of 18 CYP genes. Subsequently, genomes for Streptomyces avermitilis, with 33 CYPs, and Streptomyces peucetius, with 15 CYPs, have been reported. Although a certain number of these CYPs have known functions in secondary metabolism, as identified biochemically or through gene locus organisation, in the vast majority of Streptomyces species, CYP functions are unknown. The first detailed analysis of the CYP complement from a Streptomyces species genome has begun in the laboratories of Waterman et al. The long-term goal of this effort is to identify orphan CYP function, to establish their high resolution structure and to establish a strategy for producing novel secondary metabolites that have new biomedical function. This chapter provides an overview of CYP systems in Streptomyces species and provides a plan of how new drugs might be generated from streptomycetes by modifying the structure of specific CYPs.

Candida albicans strain NCPF 3363 was isolated from a British patient with chronic mucocutaneous candidiasis (CMC) and confirmed to be resistant to azole antifungal compounds. In this study we investigate the molecular basis of resistance and show that azole tolerance in NCPF 3363 was associated with reduced intracellular accumulation of drug and not reduced affinity for the target site, as previously indicated. Relative impermeability or the presence of transporters related to those responsible for multidrug resistance are implicated in the mechanism of resistance.

Azole antifungal compounds are important in agriculture and in the treatment of mycotic infection. The target enzyme, sterol 14 alpha-demethylase (CYP51), is inhibited through binding of triazole N-4 to the haem of this P450, as a sixth ligand together with the N-1 substituent groups interacting in some way with the apoprotein. Here we use Saccharomyces cerevisiae expression systems for the target enzyme of Candida albicans to investigate binding of enantiomers of the azole antifungal compounds SCH39304 and tetraconazole. A molecular model produced previously provided qualitative explanations for these differences. Interaction of the azole antifungal aromatic group with Phe-233 or -235 may cause the higher activity for (R)-tetraconazole while inactivity of the (SS)-enantiomer of SCH39304 was predicted to result from incompatibility of the hydrophilic sulfonyl moiety when located into the hydrophobic pocket of the active site.

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.

Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O2-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses forStreptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code 1GWI). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional β-strand transforming the five-stranded β-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactonesin vitro.

Sterol 14alpha-demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. This M. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coli yielded a P450, which, when purified to homogeneity, had the predicted molecular mass approximately 62 kDa on SDS/PAGE and bound lanosterol as a putative substrate. Sterol 14alpha-demethylase activity was shown (0.24 nmol of lanosterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general.

The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH(2)-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies.

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

The discovery of a new hemoprotein from rabbit liver in 1962 is one of the key moments in biology. Today, cytochrome P450 enzymes constitute a protein superfamily found in all domains of life and have even been described in some viruses. Herein, we describe some of the early experimental groundwork including P450 multiplicity and activity, electron transfer , and cellular localization that led to our early understanding of the molecular properties of P450 enzymes. Subsequently, following the advent of recombinant DNA technology and the development of heterologous expression systems and genomic sequencing, the traditional understanding of what actually defined a P450 was challenged: these included P450s with unusual molecular properties such as differences in electron partner proteins, fusion proteins, posttranslational modification(s) and unique catalytic activities. Selected examples from our own research findings are highlighted.

Native yeast NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) and a soluble derivative lacking 33 amino acids of the NH(2)-terminus have been overexpressed as recombinant proteins in Escherichia coli. The presence of a hexahistidine sequence at the N-terminus allowed protein purification in a single step using nickel-chelating affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the predicted molecular weights of the proteins and indicated a purity of >95%. Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Delta(22)-desaturation. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with NADPH donating electrons directly to CPR to produce a reduced intermediary form of the enzyme. The kinetic mechanism studies showed no difference between the two yeast CPRs in mechanism or after reconstitution with CYP61-mediated 22-desaturation, confirming that the retention of the NH(2)-terminable membrane anchor is functionally dispensable.

In the year 2001 a collection of yeast strains will be completed that are deleted in the 6000 open reading frames selected as putative genes by the initial bioinformatic analysis of theSaccharomyces cerevisiaegenome. The collection was produced by the transatlantic yeast gene deletion project, a collaboration involving researchers in the USA, Canada and Europe. The European effort was part of EUROFAN (European Functional Analysis Network) where some of the strains could feed into various functional analysis nodes dealing with specific areas of cell biology. With approximately 40% of human genes involved in heritable disease having a homologue in yeast and with the use of yeast in various drug discovery strategies, not least due to the dramatic increase in fungal infections, these strains will be valuable in trans-genomic studies and in specialised interest studies in individual laboratories. A detailed analysis of the project by the consortium is in preparation, here we discuss the yeast strains, reported findings and approaches to using this resource.

We report a comprehensive genetic, metabolomic, and biochemical study on the catalytic properties of Streptomyces coelicolor cytochrome P450 (P450) 154A1, known to have a unique heme orientation in its crystal structure. Deletion of the P450 154A1 gene compromised the long-term stability of the bacterial spores. A novel dipentaenone (1) with a high degree of conjugation was identified as an endogenous substrate of P450 154A1 using a metabolomics approach. The biotransformation of 1 by P450 154A1 was shown to be an unexpected intramolecular cyclization to a Paternò–Büchi-like product, without oxidation/reduction.

During the past three decades azole compounds have been developed as medical and agricultural agents to combat fungal diseases. During the 1980s they were introduced as orally active compounds in medicine and the number of such azole drugs is likely to expand in the near future. They represent a successful strategy for antifungal development, but as the incidence of fungal infection has increased coupled to prolonged use of the drugs, the (almost) inevitable emergence of resistance has occurred. This was after resistance had already been encountered as a serious problem in the field, where a larger number of azole fungicides had been employed commercially. In this review the molecular basis of how azoles work is discussed together with how fungi overcome the inhibitory effect of these compounds: through alterations in the primary target molecule (cytochrome P45051; Erg11p; sterol 14alpha-demethylase); through drug efflux mechanisms and through a suppressor mechanism allowing growth on 14-methylated sterols. Copyright 1999 Harcourt Publishers Ltd.

Cytochrome P450 has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by white-rot fungi. We have purified and reconstituted a benzo[a]pyrene hydroxylating cytochrome P450 (P450) from microsomal fractions of the white rot fungus Pleurotus pulmonarius. The microsomal P450 was recovered using a combination of n-aminooctyl agarose and hydroxyapatite chromatography and had an apparent molecular mass of 55 kDa. The purified protein exhibited moderate affinity for benzo[a]pyrene with a Ks of 66 μM calculated from the Type I substrate binding spectra produced. Reconstitution of activity was achieved and a turnover of 0.75 nmol 3-hydroxybenzo[a]pyrene product/min/nmol P450 was observed, comparable to levels of metabolism observed by animal cytochromes P450 involved in xenobiotic detoxification.

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cyto- chrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14K-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron trans- port system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disrup- tion of the gene encoding CPR. z 1999 Federation of European Biochemical Societies.

Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) is required for ergosterol biosynthesis and is the target for azole antifungal compounds. The amino acid substitution Y132H in CYP51 from clinical isolates of Candida albicans can cause fluconazole resistance by a novel change in the protein. Fluconazole binding to the mutant protein did not involve normal interaction with haem as shown by inducing a Type I spectral change. This contrasted to the wild-type protein where fluconazole inhibition was reflected in coordination to haem as a sixth ligand and where the typical Type II spectrum was obtained. The Y132H substitution occurred without drastic perturbation of the haem environment or activity allowing resistant mutants to produce ergosterol and retain fitness, an efficient strategy for resistance in nature.

The open reading frame of CYP105D1, a soluble cytochrome P450 from Streptomyces griseus, was cloned behind the tac promoter of the bacterial expression vector pSPg1910L and expressed in Escherichia coli. The recombinant protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum. CYP105D1 was active, obtaining reducing equivalents from endogenous E. coli ferredoxin and ferredoxin reductase redox partners present in E. coli. In vitro activity studies revealed CYP105D1 to catalyse the NADH- and NADPH-dependent oxidation of the xenobiotic substrates benzo[a]pyrene, erythromycin, warfarin, and testosterone. Furthermore, this activity could be stimulated in the presence of either alpha-benzoflavone or beta-benzoflavone in an analogous manner to that reported for mammalian P450 forms including human liver cytochrome P4503A4 (CYP3A4). The system produces an alternative to whole-cell biotransformation of xenobiotic for the production of drug metabolites and an experimental system for probing the structural features of a cytochrome P450 with a broad substrate range.

Human and Candida albicans CYP51 were purified to homogeneity after GAL10-based heterologous expression in yeast in order to resolve the basis for the selective inhibition of the fungal enzyme over the human orthologue by the azole drugs ketoconazole and itraconazole, used in the treatment of systemic fungal infection. The purified proteins have similar spectral characteristics, both giving a maximum at 448 nm in reduced carbon monoxide difference spectra. Substrate affinity constants of 20.8 and 29.4 microM and Vmax of 0. 15 and 0.47 nmol/min/nmol were observed for C. albicans and human enzymes, respectively, in reconstituted enzymatic assays, using an intermediate of the demethylation reaction [32-3H]-3beta-hydroxylanost-7-en-32-ol as the substrate. Both enzymes gave similar type II spectra on titration with drugs, but a reduced affinity was observed for human CYP51 using the ability of carbon monoxide to displace the drug as a ligand and by calculation of IC50. However, although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, when compared directly to CYP51 from humans, the difference was less than 10-fold. This difference is an order of magnitude lower than previously reported data based on measurements using unpurified human CYP51 enzyme preparations. Consequently, increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP51 and the potential consequences are discussed.

Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5'enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed. Here we report for the first time that glyphosate is an inhibitor of cytochrome P450 using a functional expression system for Thlaspi arvensae CYP71B1 in Escherichia coli. CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase (CPR) from Catharanthus roseus. CYP71B1 could obtain reducing equivalents in this fusion construct and metabolised the polycyclic aromatic hydrocarbon, benzo(a)pyrene. The fusion protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum. Addition of the herbicide resulted in a Type II spectrum indicative of binding via the nitrogen group to haem as a sixth ligand. A Ks of 60 microM was observed and an IC50 of 12 microM was observed for glyphosate inhibition of CYP71B1 activity. The implications of these results are discussed.

Background Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes. Methods PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS. Results Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26 μM for ketoconazole and 0.64 μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s. Conclusions Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s. General significance The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.

OBJECTIVES: In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. METHODS: Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. RESULTS: Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. CONCLUSIONS: Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

The Candida albicans sterol 14 alpha-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 mu mol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14 alpha-demethylase, 3 beta-hydroxylanost-7-en-32-al and 3 beta-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3 beta-hydroxylanost-7-en-32-al with the recombinant 14 alpha-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17 alpha-hydroxylase-17,20-lyase (P-45017 alpha,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14 alpha-demethylase is considered.