Multivariate analysis of biochar-derived carbonaceous nanomaterials for detection of heavy metal ions in aqueous systems.

This article focuses on implementing multivariate analysis to evaluate biochar-derived carbonaceous nanomaterials (BCN) from three different feedstocks for the detection and differentiation of heavy metal ions in aqueous systems. The BCN were produced from dairy manure, rice straw and sorghum straw biochar using our NanoRefinery process. The NanoRefinery process transforms biochar into advanced nanomaterials using depolymerisation/chemical oxidation and purification of nanomaterials using solvent extraction. Dairy manure biochar-derived carbonaceous nanomaterials (DMB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were utilised as probes for the evaluation of their fluorescent properties and the detection of heavy metal ions. The BCN fluorescence quenching and fluorescence recovery was tested with lead (Pb2+), nickel (Ni2+), copper (Cu2+) and mercury (Hg2+). Principal component analysis (PCA) and discriminant analysis were used to differentiate among heavy metal ions in water samples. The BCN from different feedstocks had different characteristics and produced different interactions with heavy metal ions. DMB-CN had the highest quenching for Hg2+ and Ni2+ while SSB-CN and RSB-CN responded best to Cu2+ and Pb2+, respectively. The fluorescence quenching was modelled using linear and empirical functions. PCA and discriminant analysis used the quenching measurements to differentiate heavy metal ions in aqueous system. A key result was that the discriminant analysis had a 100% accuracy to detect Pb2+, 66% for Ni2+ and Cu2+, and 33% for Hg2+. This study has shown that biochar-derived carbonaceous nanomaterials could be used in heavy metal ions sensing applications. This is the first step in the development of a fast and accurate method for the detection of heavy metal ions in waters using environmentally friendly BCN.

NanoRefinery of carbonaceous nanomaterials: Complementing dairy manure gasification and their applications in cellular imaging and heavy metal sensing.

This article describes an efficient method, combining chemical oxidation and acetone extraction, to produce carbonaceous nanomaterials from dairy manure biochar. The optical and mechanical properties are similar to methods previously reported carbonaceous nanomaterials from biomass. Our novel process cuts the processing time in half and drastically reduces the energy input required. The acetone extraction produced 10 fractions with dairy manure biochar-derived carbonaceous nanomaterials (DMB-CNs). The fraction with the carbonaceous nanomaterials, DMB-CN-E1, with highest fluorescence was selected for in-depth characterisation and for initial testing across a range of applications. DMB-CN-E1 was characterised using atomic force microscope, electrophoresis, and spectrophotometric methods. DMB-CN-E1 exhibited a lateral dimension between 11 and 28 nm, a negative charge, and excitation/emission maxima at 337/410 nm, respectively. The bioimaging potential of DMB-CN-E1 evidenced different locations and different interactions with the cellular models evaluated. DMB-CN-E1 was quenched by several heavy metal ions showing a future application of these materials in heavy metal ion detection and/or removal. The demonstrated capabilities in bioimaging and environmental sensing create the opportunity for generating added-value nanomaterials (NanoRefinery) from dairy manure biochar gasification and, thus, increasing the economic viability of gasification plants.

Microalgae biochar-derived carbon dots and their application in heavy metal sensing in aqueous systems.

This research seeks a coupled solution for managing the large amounts of biochar produced by microalgae biofuel production, and the necessity for novel, economic and accurate heavy metal sensing methods. Therefore, this study evaluated the transformation of microalgae biochar (MAB) into carbon dots (Cdots) and their subsequent application as heavy metal ion sensors in aqueous systems. The experimental phase included the transformation of MAB into microalgae biochar-derived carbon dots (MAB-Cdots), MAB-Cdot characterisation and the evaluation of the MAB-Cdots as transducers for the detection of four heavy metal ions (Pb2+, Cu2+, Cd2+, and Ni2+). MAB-Cdot fluorescence was stable over a wide range of pH and resistant to photo-bleaching, making them suitable as fluorescence probes. The MAB-Cdot fluorescence was quenched by all of the metal ions and displayed different quenching levels. Depending upon the ions involved, MAB-Cdots were used to detect the presence of heavy metal ions from concentrations of 0.012 µM up to 2 mM by measuring the reduction in fluorescence intensity. Neutral and slightly alkaline pHs were optimal for Cu2+ Ni2+ and Pb2+ heavy metal quenching. To quantify the concentration of the heavy metal ions, linear and logarithmic functions were used to model the MAB-Cdot fluorescence quenching. The sensing mechanism was determined to be reversible and purely collisional with some fluorophores less accessible than the others. This work demonstrated the ability to produce Cdots from microalgae biochar, examined their application as a transducer for detecting heavy metal ions in aqueous systems and paves the way for novel sensing systems using MAB-Cdots.

Does percutaneous tibial nerve stimulation improve global pelvic function in women with faecal incontinence?

AIM: Percutaneous tibial nerve stimulation (PTNS) is a minimally invasive treatment for faecal incontinence. Many patients with faecal incontinence have coexisting pelvic floor disorders such as urinary incontinence and vaginal symptoms. We utilized a pelvic floor assessment tool to analyse any effect of PTNS on global pelvic floor function. METHODS: Patients with faecal incontinence attending our institution who had failed to respond sufficiently to biofeedback were offered a course of PTNS. Patients underwent pre- and post-stimulation assessment with a validated electronic Personal Assessment Questionnaire - Pelvic Floor (ePAQ-PF) for pelvic floor disorders. Scores were compared to assess the effect of treatment on global pelvic floor function. RESULTS: During the study period pre- and post-stimulation ePAQ-PF data were available for 60 patients (55% of all patients starting PTNS). In this cohort there was a significant improvement in bowel continence, bowel related quality of life, irritable bowel syndrome and bowel evacuation with a large effect size for continence and bowel related quality of life. There was also a significant improvement in non-bowel related symptoms, including urinary pain and stress incontinence, urinary related quality of life and bowel related sexual function. Sixty-five per cent of those who answered the question reported improvement in global health after stimulation. CONCLUSION: For patients presenting with faecal incontinence, PTNS appears to have a positive effect on bowel related function in approximately two-thirds of patients. However, for treatment responders, improvement appears to relate mainly to improvement in bowel related function rather than a global pelvic floor effect.

The clinical candidate VT-1161 is a highly potent inhibitor of Candida albicans CYP51 but fails to bind the human enzyme.

The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [Kd], ≤ 39 nM) and similar to that of the pharmaceutical azole antifungals (Kd, ≤ 50 nM). In stark contrast, VT-1161 at concentrations up to 86 µM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 µM). In addition, VT-1161 (MIC = 0.002 µg ml(-1)) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 µg ml(-1)). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.

Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

Azole resistance by loss of function of the sterol Δ5,6-desaturase gene (ERG3) in Candida albicans does not necessarily decrease virulence.

The inactivation of ERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.

Triclosan antagonizes fluconazole activity against Candida albicans.

Triclosan is a broad-spectrum antimicrobial compound commonly used in oral hygiene products. Investigation of its activity against Candida albicans showed that triclosan was fungicidal at concentrations of 16 mg/L. However, at subinhibitory concentrations (0.5-2 mg/L), triclosan antagonized the activity of fluconazole. Although triclosan induced CDR1 expression in C. albicans, antagonism was still observed in cdr1Δ and cdr2Δ strains. Triclosan did not affect fluconazole uptake or alter total membrane sterol content, but did induce the expression of FAS1 and FAS2, indicating that its mode of action may involve inhibition of fatty acid synthesis, as it does in prokaryotes. However, FAS2 mutants did not exhibit increased susceptibility to triclosan, and overexpression of both FAS1 and FAS2 alleles did not alter triclosan susceptibility. Unexpectedly, the antagonistic effect was specific for C. albicans under hypha-inducing conditions and was absent in the non-filamentous efg1Δ strain. This antagonism may be due to the membranotropic activity of triclosan and the unique composition of hyphal membranes.

Expression of bacterial levanase in yeast enables simultaneous saccharification and fermentation of grass juice to bioethanol.

This study demonstrates use of recombinant yeast to simultaneously saccharify and ferment grass juice (GJ) to bioethanol. A modified Bacillus subtilis levanase gene (sacC) in which the native bacterial signal sequence was replaced with a yeast α-factor domain, was synthesised with yeast codon preferences and transformed into Saccharomyces cerevisiae (strain AH22) using the expression vector pMA91. AH22:psacC transformants secreted sacCp as an active, hyper-glycosylated (>180 kDa) protein allowing them to utilise inulin (ß[2-1] linked fructose) and levan (ß[2-6] linkages) as growth substrates. The control (AH22:pMA91) strain, transformed with empty plasmid DNA was not able to utilise inulin or levan. When cultured on untreated GJ levels of growth and bioethanol production were significantly higher in experiments with AH22:psacC than with AH22:pMA91. Bioethanol yields from AH22:psacC grown on GJ (32.7[±4] mg mL(-1)) compared closely to those recently achieved (Martel et al., 2010) using enzymatically pre-hydrolysed GJ (36.8[±4] mg mL(-1)).

Heterologous expression of mutated eburicol 14alpha-demethylase (CYP51) proteins of Mycosphaerella graminicola to assess effects on azole fungicide sensitivity and intrinsic protein function.

The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14alpha-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO(7)-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion DeltaY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.

Expression, purification and use of the soluble domain of Lactobacillus paracasei beta-fructosidase to optimise production of bioethanol from grass fructans.

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.

Differential azole antifungal efficacies contrasted using a Saccharomyces cerevisiae strain humanized for sterol 14 alpha-demethylase at the homologous locus.

Inhibition of sterol-14 alpha-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14 alpha-methyl-ergosta-8, 24(28)-dien-3beta-6 alpha-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.

CYP56 (Dit2p) in Candida albicans: characterization and investigation of its role in growth and antifungal drug susceptibility.

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Cytochrome P450 biodiversity and biotechnology.

CYP (cytochrome P450) biodiversity and biotechnology is of importance given the industrial applications and potential for the huge array of genes and proteins that can constitute up to 1% of a coding genome. Historical biotechnological roles for CYPs in mutant fungi diverting the flux of metabolites towards penicillin production, in biotransformations allowing the production of corticosteroids and CYPs as drug targets contribute to interest in the roles of orphan CYPs in the emerging genomes. This area includes studies related to biotransformations and bioremediation, natural product synthesis and its manipulation, tools for exploiting CYPs and using CYPs as biomarkers and drug targets. Fundamental studies on diverse structure and function, on the ecological and evolution of CYPs through geological time and in drug/pesticide resistance also contribute distinctively to this field of CYP research.

Towards engineered topogenesis of cytochrome b(5) and P450 for in vivo transformation of xenobiotics.

Nature is endowed with catalysts capable of an unprecedented diversity of biotransformations, beyond the capabilities of synthetic chemistries. In a biotechnological context, there is a growing and emerging need to tap this catalytic potential. CYP (cytochrome P450) represents a superfamily of enzymes capable of a diverse array of catalytic activities. Distinct members are engaged in biosynthetic reactions within many organisms, while others have a role in the detoxification of foreign compounds. The latter substrates include medicines, pollutants, pesticides, carcinogens, perfumes and herbicides, representing considerable applied importance for pharmacology and toxicology. CYPs show a high degree of stereo- and regio-specificity for their reactions, which have wide industrial applications. Recombinant CYPs are commonly expressed as active recombinant cytosolic forms in Escherichia coli. However, selective permeability of E. coli to many substrates and products can cause problems with product recovery when using whole-cell systems. To overcome these problems, we have been developing approaches to facilitate export of functional recombinant haemoproteins to the inner membrane, periplasm and the outer membrane of E. coli. Here, we describe the progress in relation to cytochrome b(5) and CYPs.

Cytochrome b(5) modulation of 17{alpha} hydroxylase and 17-20 lyase (CYP17) activities in steroidogenesis.

CYP17 is a steroidogenic enzyme located in the zona fasciculata and zona reticularis of the adrenal cortex and gonad tissues and which has dual functions - hydroxylation and as a lyase. The first activity gives hydroxylation of pregnenolone and progesterone at the C(17) position to generate 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone, while the second enzymic activity cleaves the C(17)-C(20) bond of 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone to form dehydroepiandro-sterone and androstenedione respectively. The modulation of these two activities occurs through cytochrome b(5). Association of cytochrome b(5) and CYP17 is thought to be based primarily on electrostatic interactions in which the negatively charged residues pair up with positively charged residues on the proximal surface of the CYP17 molecule. Non-specific interactions of the hydrophobic membrane regions of cytochrome b(5) and CYP17 are also thought to play a crucial role in the association of these two haemoproteins. Although cytochrome b(5) is known to stimulate CYP activity by contributing the second electron in the catalytic cycle, in the case of CYP17, the mechanism of cleavage stimulation proceeds via an allosteric mode. It is hypothesised that cytochrome b(5) promotes the cleavage by aligning the iron-oxygen complex attack onto the C(20) rather than the C(17) atom of the steroid substrate molecule. Thus, further understanding of the mechanism of modulation by cytochrome b(5) of the hydroxylase and lyase activities should shed new insights on developing therapeutic targets in CYP17-linked biochemical processes such as adrenarche, polycystic ovary syndrome and prostate cancer.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation.

AIMS: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. METHODS AND RESULTS: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K(d) and relative B(max) values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K(d) and relative B(max), which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. CONCLUSIONS: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. SIGNIFICANCE AND IMPACT OF THE STUDY: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans.

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.

Human sterol 14alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5).

Human sterol 14alpha-demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway. The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalytic cycle. Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51. The cytochrome b(5) derivatives were genetically engineered forms of the native rat cytochrome b(5) core-tail: the soluble globular b(5) core (core), the core linked at its N-terminus with the secretory signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail). The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active. In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction. Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting electrons from soluble yeast CPR and in its ability to reduce P450. We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core. This is the first report showing the stimulation of CYP51 by cytochrome b(5).

Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase.

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.