Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1

Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the “soft” recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1–22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.     

Binding of a Cyano- and Fluoro-containing Drug Bicalutamide to Cytochrome P450 46A1: UNUSUAL FEATURES AND SPECTRAL RESPONSE*

Cytochrome P450 46A1 (CYP46A1) is the cholesterol 24-hydroxylase initiating the major pathways of cholesterol removal from the brain, and bicalutamide (BIC) is a drug of choice for the treatment of progressive androgen-dependent prostate cancer. We evaluated the interactions of BIC with CYP46A1 by x-ray crystallography and by conducting solution and mutagenesis studies. Because BIC is administered to patients as a racemic mixture of the S and R isomers, we studied all three, racemic BIC as well as the S and R isomers. Co-crystallization of CYP46A1 with racemic BIC led to structure determinations at 2.1 Å resolution with the drug complexes exhibiting novel properties. Both BIC isomers bind to the CYP46A1 active site in very similar single orientation and adopt an energetically unfavorable folded conformation. This folded BIC conformation is correlated with drug-induced localized shifts of amino acid side chains in CYP46A1 and unusual interactions in the CYP46A1-BIC complex. One of these interactions involves a water molecule that is coordinated to the P450 heme iron and also hydrogen-bonded to the BIC nitrile. Due to polarization of the water in this environment, the heme elicits previously unreported types of P450 spectral responses. We also observed that access to the P450 active site was affected by differential recognition of S versus R isomers at the CYP46A1 surface arising from BIC conformational polymorphism.     

Structural and biochemical characterization of Mycobacterium tuberculosis CYP142: evidence for multiple cholesterol 27-hydroxylase activities in a human pathogen

The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å, revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ΔCYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism.     

The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546–9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.     

Crystal structure of H2O2-dependent cytochrome P450SPalpha with its bound fatty acid substrate: insight into the regioselective hydroxylation of fatty acids at the alpha position

Cytochrome P450_SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H₂O₂-dependent P450. P450_SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450_SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the C_α of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450_SPα. Mutations at the active site and the F–G loop of P450_SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450_BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450_SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450_BSβ.     

In vivo consequences of cholesterol-24S-hydroxylase (CYP46A1) inhibition by voriconazole on cholesterol homeostasis and function in the rat retina

Cholesterol 24S-hydroxylase (CYP46A1) converts cholesterol into 24S-hydroxycholesterol in neurons and participates in cholesterol homeostasis in the central nervous system, including the retina. We aimed to evaluate the consequences of CYP46A1 inhibition by voriconazole on cholesterol homeostasis and function in the retina. Rats received daily intraperitoneal injections of voriconazole (60 mg/kg), minocycline (22 mg/kg), voriconazole plus minocycline, or vehicle during five consecutive days. The rats were submitted to electroretinography to monitor retinal functionality. Cholesterol and 24S-hydroxycholesterol were measured in plasma, brain and retina by gas chromatography-mass spectrometry. The expression of CYP46A1, and GFAP as a marker for glial activation was analyzed in the retina and brain. Cytokines and chemokines were measured in plasma, vitreous, retina and brain. Voriconazole significantly impaired the functioning of the retina as exemplified by the reduced amplitude and increased latency of the b-wave of the electroretinogram, and altered oscillary potentials. Voriconazole decreased 24S-hydroxycholesterol levels in the retina. Unexpectedly, CYP46A1 and GFAP expression was increased in the retina of voriconazole-treated rats. ICAM-1 and MCP-1 showed significant increases in the retina and vitreous body. Minocycline did not reverse the effects of voriconazole. Our data highlighted the cross talk between retinal ganglion cells and glial cells in the retina, suggesting that reduced 24S-hydroxycholesterol concentration in the retina may be detected by glial cells, which were consequently activated.     

Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn²⁰² in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b₅ might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.     

Pharmacologic Stimulation of Cytochrome P450 46A1 and Cerebral Cholesterol Turnover in Mice

Cytochrome P450 46A1 (CYP46A1) is a brain-specific cholesterol 24-hydroxylase responsible for the majority of cholesterol elimination from the brain. Genetically increased CYP46A1 expression in mice leads to improved cognition and decreases manifestations of Alzheimer disease. We found that four pharmaceuticals (efavirenz (EFV), acetaminophen, mirtazapine, and galantamine) prescribed for indications unrelated to cholesterol maintenance increased CYP46A1 activity in vitro. We then evaluated the anti-HIV medication EFV for the mode of interaction with CYP46A1 and the effect on mice. We propose a model for CYP46A1 activation by EFV and show that EFV enhanced CYP46A1 activity and cerebral cholesterol turnover in animals with no effect on the levels of brain cholesterol. The doses of EFV administered to mice and required for the stimulation of their cerebral cholesterol turnover are a hundred times lower than those prescribed to HIV patients. At such small doses, EFV may be devoid of adverse effects elicited by high drug concentrations. CYP46A1 could be a novel therapeutic target and a tool to further investigate the physiological and medical significance of cerebral cholesterol turnover.     

Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 Å) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 Å). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 α-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an α-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.     

Human Cytochrome P450 2E1 Structures with Fatty Acid Analogs Reveal a Previously Unobserved Binding Mode

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9–C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with ω-imidazolyl-octanoic fatty acid, ω-imidazolyl-decanoic fatty acid, and ω-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the ω-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe²⁹⁸ side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B′ helices at successive distances from the active site.     

Antifungal drug resistance pattern of Candida. spp isolated from vaginitis in Ilam-Iran during 2013-2014

Vaginal Candidiasis is the most common and important opportunistic fungal infection in women. By increasing use of antifungal drugs in recent years, it has caused drug resistance. This study aims to evaluate antifungal drugs susceptibility of Candida. spp isolated of women with vaginitis from Ilam-Iran during 2013-2014. samples were collected and cultured from 385 women with vaginitis, then Candida.spp was diagnosed by standard method. Antifungal drug susceptibility test for nystatin 100 unit/disk, fluconazole 10µg/disk, itraconazole 10µg/disk, ketoconazole 10µg/disk, amphotericinB 20µg/disk, clotrimazole 10µg/disk, posaconazole 5µg/disk, and voriconazole 1µg/disk were carried out by M44-A method(CLSI). From all culture positive samples, 150 isolates were Candida albicans and 89 isolates were non-albicans. The resistance to fluconazole, itraconazole, ketoconazole, clotrimazole, voriconazole, posaconazole, nystatin and amphotericin B was 76%, 62%, 72%, 55%, 6%, 7%, 1% and 0%. The highest resistance was seen for fluconazole , itraconazole, and the highest susceptible was seen for nystatin and amphotericin B. These results indicate nystatin and amphotericin B can be used as the first line for empirical therapy of vaginal candidiasis in the district.     

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1′, respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B′ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.     

Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.     

Retinoic Acid-related Orphan Receptor α Regulates Diurnal Rhythm and Fasting Induction of Sterol 12α-Hydroxylase in Bile Acid Synthesis

Sterol 12α-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor α (RORα). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting increased RORα protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORα to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12α-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORα response element in the CYP8B1 promoter. RORα recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORα is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.     

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Polymorphisms of CYP51A1 from Cholesterol Synthesis: Associations with Birth Weight and Maternal Lipid Levels and Impact on CYP51 Protein Structure

We investigated the housekeeping cytochrome P450 CYP51A1 encoding lanosterol 14α-demethylase from cholesterol synthesis that was so far not directly linked to human disorders. By direct sequencing of CYP51A1 in 188 women with spontaneous preterm delivery and 188 unrelated preterm infants (gestational age <37 weeks) we identified 22 variants where 10 are novel and rare. In infants there were two novel CYP51A1 variants where damaging effects of p.Tyr145Asp from the substrate recognition region, but not p.Asn193Asp, were predicted by PolyPhen2 and SIFT. This was confirmed by molecular modeling showing that Tyr145Asp substitution results in changed electrostatic potential of the CYP51 protein surface and lengthened distance to the heme which prevents hydrogen bonding. The CYP51 Tyr145Asp mutation is rare and thus very interesting for further structure/function relationship studies. From the 12 identified known variants rs6465348 was chosen for family based association studies due to its high minor allele frequency. Interestingly, this CYP51A1 common variant associates with small for gestational age weight in newborns (p = 0.028) and lower blood total cholesterol and low density lipoprotein cholesterol levels in mothers in 2nd trimester of pregnancy (p = 0.042 and p = 0.046 respectively). Our results indicate a new link between a cholesterol synthesis gene CYP51A1 and pregnancy pathologies.     

Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Synthesis and pharmacokinetic study of a 11C-labeled cholesterol 24-hydroxylase inhibitor using ‘in-loop’ [11C]CO2 fixation method

Cholesterol 24-hydroxylase, also known as CYP46A1 (EC 1.14.13.98), is a monooxygenase and a member of the cytochrome P450 family. CYP46A1 is specifically expressed in the brain where it controls cholesterol elimination by producing 24S-hydroxylcholesterol (24-HC) as the major metabolite. Modulation of CYP46A1 activity may affect Aβ deposition and p-tau accumulation by changing 24-HC formation, which thereafter serves as potential therapeutic pathway for Alzheimer’s disease. In this work, we showcase the efficient synthesis and preliminary pharmacokinetic evaluation of a novel cholesterol 24-hydroxylase inhibitor 1 for use in positron emission tomography.     

Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.