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Discusses the separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach. Formation of cysteine from O-acetylserine and bisulphide; Incorporation of inorganic sulphur into the amino acid cysteine; Transport of cysteine between subcellular compartments.

The cytochrome P450 CYP168A1 from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli followed by purification and characterization of function. CYP168A1 is a fatty acid hydroxylase that hydroxylates saturated fatty acids, including myristic (0.30 min-1), palmitic (1.61 min-1) and stearic acids (1.24 min-1), at both the ω-1- and ω-2-positions. However, CYP168A1 only hydroxylates unsaturated fatty acids, including palmitoleic (0.38 min-1), oleic (1.28 min-1) and linoleic acids (0.35 min-1), at the ω-1-position. CYP168A1 exhibited a catalytic preference for palmitic, oleic and stearic acids as substrates in keeping with the phosphatidylcholine-rich environment deep in the lung that is colonized by P. aeruginosa., Competing Interests: The authors have declared that no competing interests exist.

Here we report the first evaluation of isavuconazole inhibition of Aspergillus fumigatus CYP51 and thus sterol biosynthesis in the fungus. Voriconazole and isavuconazole both bound tightly to recombinant A. fumigatus CYP51 isoenzymes A and B (AfCYP51A and AfCYP51B) isolated in Escherichia coli membranes. CYP51 reconstitution assays confirmed that AfCYP51A and AfCYP51B as well as three AfCYP51A mutants known to confer azole resistance (G54W, L98H and M220K) were strongly inhibited by both triazoles. Voriconazole bound relatively weakly to purified Homo sapiens CYP51 (HsCYP51), unlike isavuconazole that bound tightly. However, isavuconazole was a relatively poor inhibitor of HsCYP51 activity, with an IC 50 value (half-maximal inhibitory concentration) of 25 µM, which was 55- to 120-fold greater than those observed for the A. fumigatus CYP51 enzymes, albeit not as poor an inhibitor of HsCYP51 as voriconazole with an IC 50 value of 112 µM. Sterol analysis of triazole-treated A. fumigatus Af293 cells confirmed that isavuconazole and voriconazole both inhibited cellular CYP51 activity with the accumulation of 14-methylated sterol substrates and depletion of ergosterol levels. Isavuconazole elicited a stronger perturbation of the sterol composition in A. fumigatus Af293 than voriconazole at 0.0125 µg/mL, indicating increased potency. However, complementation studies in Saccharomyces cerevisiae using strains containing AfCYP51A and AfCYP51B showed isavuconazole to be equally as effective at inhibiting CYP51 activity as voriconazole. These in vitro studies suggest that isavuconazole is an effective alternative to voriconazole as an antifungal agent against the target CYP51 in A. fumigatus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆ 8,14 -sterol (<95%). The combination of [ 2 H 3 -methyl]methionine incubation to intact cultures showing C 28 -ergosterol incorporates 2- 2 H atoms and C 29 -7-dehydroporiferasterol incorporates 5 2 H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ∆ 24(28) - and ∆ 25(27) -olefin pathways to C 28 - and C 29 -sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C 28 /C 29 -∆ 5,7 -sterols, viable encysted cells (mature cyst) possess mostly C 29 -∆ 5 -sterol and non-viable encysted cells possess C 28 /C 29 -∆ 5,7 -sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ∆ 5,7 -sterol biosynthesis (to ∆ 5,7 ) - metabolism (to ∆ 5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates ( n = 96) with a MIC range of 0.016 to 0.25 μg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 μg/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 μg/ml), especially Aspergillus terreus , Trichophyton rubrum , Candida albicans , Candida glabrata , Candida krusei , Cryptococcus gattii , Cryptococcus neoformans , and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 μg/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo , once-daily intranasal administration of PC1244 (3.2 to 80 μg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans., (Copyright © 2018 Colley et al.)

Background: Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice., Results: Genes encoding the 11α-steroid hydroxylase enzymes from Aspergillus ochraceus (11α-SH Aoch ) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SH Aoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 µM progesterone) producing either 11α-hydroxyprogesterone as the sole metabolite (AH22:p11α-SH Aoch ) or a 7:1 mixture of 11α-hydroxyprogesterone and 6β-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SH Aoch and AH22:p509C12 were comparable resulting in ≥75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11α-SH Aoch gene into the yeast genome (AH22:11α-SHAoch+K) resulted in a 36% reduction in yield of 11α-hydroxyprogesterone to 174 µmol/L using 300 µM progesterone. However, increasing progesterone concentration to 955 µM and optimizing growth conditions increased 11α-hydroxyprogesterone production to 592 µmol/L product formed., Conclusions: The progesterone 11α-steroid hydroxylases from A. ochraceus and R. oryzae , both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae . It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11α-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.

Prior to characterization of antifungal inhibitors that target CYP51, Trichophyton rubrum CYP51 was expressed in Escherichia coli , purified, and characterized. T. rubrum CYP51 bound lanosterol, obtusifoliol, and eburicol with similar affinities (dissociation constant [ K d ] values, 22.7, 20.3, and 20.9 μM, respectively) but displayed substrate specificity, insofar as only eburicol was demethylated in CYP51 reconstitution assays (turnover number, 1.55 min -1 ; K m value, 2 μM). The investigational agent VT-1161 bound tightly to T. rubrum CYP51 ( K d = 242 nM) with an affinity similar to that of clotrimazole, fluconazole, ketoconazole, and voriconazole ( K d values, 179, 173, 312, and 304 nM, respectively) and with an affinity lower than that of itraconazole ( K d = 53 nM). Determinations of 50% inhibitory concentrations (IC 50 s) using 0.5 μM CYP51 showed that VT-1161 was a tight-binding inhibitor of T. rubrum CYP51 activity, yielding an IC 50 of 0.14 μM, whereas itraconazole, fluconazole, and ketoconazole had IC 50 s of 0.26, 0.4, and 0.6 μM, respectively. When the activity of VT-1161 was tested against 34 clinical isolates, VT-1161 was a potent inhibitor of T. rubrum growth, with MIC 50 , MIC 90 , and geometric mean MIC values of ≤0.03, 0.06, and 0.033 μg ml -1 , respectively. With its selectivity versus human CYP51 and drug-metabolizing cytochrome P450s having already been established, VT-1161 should prove to be safe and effective in combating T. rubrum infections in patients., (Copyright © 2017 American Society for Microbiology.)

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC 50 s], 0.23 μM and 0.22 μM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 μg/ml, while those of voriconazole ranged from 0.064 to 4 μg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC 50 (determined based on optical density [OD]) values of 0.0012 to 0.034 μg/ml, whereas voriconazole (0.019 to >1 μg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 μg/ml), including Aspergillus terreus , Trichophyton rubrum , Candida albicans , Candida glabrata , Candida krusei , Cryptococcus gattii , Cryptococcus neoformans , and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 μg/mouse, while posaconazole showed similar effects (44%) at 14 μg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung., (Copyright © 2017 Colley et al.)