Identification and Expression Analysis of Cytochrome P450 Genes Probably Involved in Triterpenoid Saponins Biosynthesis in Astragalus mongholicus.

Cytochromes P450 (P450s) are one of the largest enzymatic protein families and play critical roles in the synthesis and metabolism of plant secondary metabolites. Astragaloside IV (AS-IV) is one of the primary active components in Astragalus herbs, exhibiting diverse biological activities and pharmacological effects. However, P450s involved in the astragaloside biosynthesis have not been systematically analyzed in Astragalus mongholicus (A. mongholicus). In this study, we identified 209 P450 genes from the genome of A. mongholicus (AmP450s), which were classified into nine clans and 47 families and performed a systematic overview of their physical and chemical properties, phylogeny, gene structures and conserved motifs. Weighted gene co-expression network analysis (WGCNA) revealed that AmP450s are critical in the astragaloside biosynthesis pathway. The expression levels of these AmP450s were verified by quantitative real-time PCR (qRT-PCR) analysis in the root, stem and leaf, showing that most AmP450s are abundant in the root. Additionally, the correlation analysis between gene expressions and AS-IV content showed that twelve AmP450s, especially CYP71A28, CYP71D16 and CYP72A69, may have significant potential in the biosynthesis of astragaloside. This study systematically investigates the P450s of A. mongholicus and offers valuable insights into further exploring the functions of CYP450s in the astragaloside biosynthesis pathway.

Intraplantar aminoglutethimide, a P450scc inhibitor, reduced the induction of mechanical allodynia in a rat model of thrombus-induced ischemic pain.

Neuroactive steroids (NASs) directly affect neuronal excitability. Despite their role in the nervous system is intimately linked to pain control, knowledge is currently limited. This study investigates the peripheral involvement of NASs in chronic ischemic pain by targeting the cytochrome P450 side-chain cleavage enzyme (P450scc). Using a rat model of hind limb thrombus-induced ischemic pain (TIIP), we observed an increase in P450scc expression in the ischemic hind paw skin. Inhibiting P450scc with intraplantar aminoglutethimide (AMG) administration from post-operative day 0 to 3 significantly reduced the development of mechanical allodynia. However, AMG administration from post-operative day 3 to 6 did not affect established mechanical allodynia. In addition, we explored the role of the peripheral sigma-1 receptor (Sig-1R) by co-administering PRE-084 (PRE), a Sig-1R agonist, with AMG. PRE reversed the analgesic effects of AMG during the induction phase. These findings indicate that inhibiting steroidogenesis with AMG alleviates peripheral ischemic pain during the induction phase via Sig-1Rs.

[Research progress in biosynthesis-related enzymes of coumarin compounds and their bioactivities].

Coumarins are natural products with benzopyran ring as the parent nucleus. Numerous coumarin derivatives exhibit a variety of pharmacological activities, including antibacterial, anti-inflammatory, antitumor, anti-coagulant, anti-osteoporotic, and insecticidal activities. Therefore, they play an important role in both medicine and agriculture. The development and utilization of coumarin derivatives have attracted increasing attention. The advancement of gene sequencing technology and the rapid progress in synthetic bio-logy have led to significant advancement in the biosynthesis of coumarin derivatives, and has received increasing attention from global researchers. This paper presents a comprehensive overview of the key biosynthesis-related enzymes of coumarin derivatives, such as cytochrome P450 enzyme(CYP450), prenyltransferase(PT), UDP-glucosyltransferase(UGT). Additionally, the pharmacological activities of these enzymes, including anti-tumor, anti-inflammatory, antioxidant, and antibacterial activities, are systematically summarized. This review aims to provide a valuable reference for the biosynthesis of coumarin derivatives and further exploration of their medicinal potential.

Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y456 differ from that of AgCPR-His456. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Chlorantraniliprole Resistance in Spodoptera frugiperda: Resistance Monitoring, Resistance Risk, and Resistance Mechanisms.

Spodoptera frugiperda is a significant global pest, and chlorantraniliprole (CAP) is extensively used in China for its control. Understanding CAP resistance in S. frugiperda is crucial for effective management of this pest. Field populations exhibited varying degrees of resistance to CAP (RR = 1.74-5.60-fold). After 10 generations of selection, the CAP-resistant strain developed over 10-fold resistance, with a realized heritability (h2) of 0.10. Genetic analysis reveals inheritance patterns as autosomal, incomplete recessive, and monofactorial. The CAP-resistant strain showed limited cross-resistance to lufenuron and tetrachlorantraniliprole, negative cross-resistance to spinetoram, and no observed cross-resistance to other insecticides. Biochemical analysis suggested that P450-mediated detoxification is the primary resistance mechanism, with 26 genes overexpressed in the CAP-resistant strain. Additionally, the knockdown of CYP4L13, CYP6B39, CYP6B40, and CYP4G74 significantly increased the sensitivity of the resistant larvae to CAP. These findings highlight the resistance risk of CAP in S. frugiperda and emphasize the crucial role of P450 enzymes in resistance.

Potential Involvement of MnCYP710A11 in Botrytis cinerea Resistance in Arabidopsis thaliana and Morus notabilis.

Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene (MnCYP710A11) was discovered in mulberry (Morus notabilis). Bioinformatic analysis and expression pattern analysis were conducted to elucidate the involvement of MnCYP710A11 in combating Botrytis cinerea infection. After the infection of B. cinerea, there was a notable increase in the expression of MnCYP710A11. MnCYP710A11 is overexpressed in Arabidopsis and mulberry and strongly reacts to B. cinerea. The overexpression of the MnCYP710A11 gene in Arabidopsis and mulberry led to a substantial enhancement in resistance against B. cinerea, elevated catalase (CAT) activity, increased proline content, and reduced malondialdehyde (MDA) levels. At the same time, H2O2 and O2- levels in MnCYP710A11 transgenic Arabidopsis were decreased, which reduced the damage of ROS accumulation to plants. Furthermore, our research indicates the potential involvement of MnCYP710A11 in B. cinerea resistance through the modulation of other resistance-related genes. These findings establish a crucial foundation for gaining deeper insights into the role of cytochrome P450 in mulberry plants.

Molecular insights into the functional analysis of P450 CYP321A7 gene in the involvement of detoxification of lambda-cyhalothrin in Spodoptera frugiperda.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is a widely recognized global agricultural pest that has significantly reduced crop yields all over the world. S. frugiperda has developed resistance to various insecticides. Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides, leading to increased resistance in insect populations. However, the function of the specific P450 gene for lambda-cyhalothrin resistance in S. frugiperda was unclear. Herein, the expression patterns of 40 P450 genes in the susceptible and lambda-cyhalothrin-resistant populations were analyzed. Among them, CYP321A7 was found to be overexpressed in the resistant population, specifically LRS (resistance ratio = 25.38-fold) derived from a lambda-cyhalothrin-susceptible (SS) population and FLRS (a population caught from a field, resistance ratio = 63.80-fold). Elevated enzyme activity of cytochrome P450 monooxygenases (P450s) was observed for LRS (2.76-fold) and the FLRS (4.88-fold) as compared to SS, while no significant differences were observed in the activities of glutathione S-transferases and esterases. Furthermore, the knockdown of CYP321A7 gene by RNA interference significantly increased the susceptibility to lambda-cyhalothrin. Remarkably, the knockdown of CYP321A7 reduced the enzymatic activity of P450 by 43.7%, 31.9%, and 22.5% in SS, LRS, and FLRS populations, respectively. Interestingly, fourth-instar larvae treated with lambda-cyhalothrin at the LC30 dosage had a greater mortality rate due to RNA interference-induced suppression of CYP321A7 (with increases of 61.1%, 50.0%, and 45.6% for SS, LRS, and FLRS populations, respectively). These findings suggest a link between lambda-cyhalothrin resistance and continual overexpression of CYP321A7 in S. frugiperda larvae, emphasizing the possible importance of CYP321A7 in lambda-cyhalothrin detoxification in S. frugiperda.

Baseline of susceptibility, risk assessment, biochemical mechanism, and fitness cost of resistance to dimpropyridaz, a novel pyridazine pyrazolecarboxamide insecticide, in Bemisia tabaci from China.

Bemisia tabaci is one of the most destructive agricultural insect pests around the world, and it has developed high levels of resistance to most pesticides. Dimpropyridaz, a novel insecticide developed by BASF, displays excellent activity against piercing-sucking insect pests. In this study, baseline of susceptibility showed all tested field populations of B. tabaci are susceptible to dimpropyridaz. After continuous selection with dimpropyridaz in the lab, a B. tabaci strain (F12) developed 2.2-fold higher level of resistance compared with a susceptible MED-S strain, and the realized heritability (h2) was estimated as 0.0518. The F12 strain displayed little cross-resistance to afidopyropen, cyantraniliprole, sulfoxaflor, or abamectin, and significantly increased activity of cytochrome P450 monooxygenase (P450). The fitness cost of dimpropyridaz resistance was evident in F12 strain, which had a relative fitness of 0.95 and significantly lower fecundity per female compared with MED-S strain. Taken together, B. tabaci displays high susceptibility to dimpropyridaz in the field, and low risk of developing resistance to dimpropyridaz under successive selection pressure. Little cross-resistance to popular insecticides was found, and fitness cost associated dimpropyridaz resistance was observed. Higher activity of cytochrome P450 in the F12 strain, may be involved in the process of detoxifying dimpropyridaz in whitefly.

The cytochrome P450 gene CYP4g1 driven by high temperature confers abamectin tolerance on Liriomyza trifolii through promoting cuticular hydrocarbons biosynthesis.

Liriomyza trifolii, an invasive pest, poses a substantial threat to horticultural and vegetable plants. It spreads rapidly, especially in hot weather, leading to large-scale outbreaks with strong thermotolerance and insecticide resistance. In this study, mortality and LtCYP4g1 expression in L. trifolii were evaluated after thermal and insecticides exposure. Furthermore, functional verification of LtCYP4g1 was conducted through RNA interference and bacterial survival assays in Escherichia coli containing recombinant LtCYP4g1 protein. Results indicated that a short time exposure to high temperature incresed insecticide tolerance of L. trifolii, attributed to decreased mortality and induced LtCYP4g1 expression; LtCYP4g1 was involved in stimulating synthesis of cuticular hydrocarbons (CHCs) and elevating epicuticle lipid content and thickness, and E. coli cells overexpressing LtCYP4g1 exhibited significant tolerance to thermal and insecticide stress. In general, P450-mediated tolerance of L. trifolii was enhanced by high temperature, with LtCYP4g1 playing a role in promoting biosynthesis of CHCs for thickening epidermal lipid barrier and reducing cuticular penetration. This study provides a framework for delving into the function of CYP450s in insecticide detoxification and illustrates the role of global warming in driving the evolution of L. trifolii.

A down-regulated cytochrome P450 in Neoseiulus barkeri Hughes (Acari: Phytoseiidae) can dechlorinate and hydroxylate chlorpyrifos without producing chlorpyrifos-oxon.

Selection of chemical-resistant predatory mites is a good alternative to balance the contradiction between chemical control and biological control. Previously, a resistant strain of Neoseiulus barkeri for chlorpyrifos was obtained. In the current study, two up-regulated (NbCYP3A6, NbCYP3A16) and one down-regulated (NbCYP3A24) P450s were screened through differential expression analysis and other detoxification-related genes such as CCEs, GST, etc. were not found. 3D modelling and molecular docking indicated that the chlorine at position 5 on the pyridine ring of chlorpyrifos, as well as a methyl group, were closest to the heme iron of the enzymes (less than 5 Å). Three active recombinant P450 proteins were heterologously expressed and metabolized with chlorpyrifos in vitro. HPLC assay showed that only NbCYP3A24 could metabolize chlorpyrifos, with a metabolism rate of 21.60 %. Analysis of the m/z of metabolites by LC-MS/MS showed that chlorine at the 5C position of chlorpyrifos was stripped and hydroxylated, whereas chlorpyrifos-oxon, a common product of oxidation by P450, was not found. Knockdown of the NbCYP3A24 gene in the susceptiblestrain did reduce the susceptibility of N. barkeri to chlorpyrifos, suggesting that the biological activity of the metabolite may be similar to chlorpyrifos-oxon, thus enhancing the inhibitory effect on the target.

Total Synthesis Facilitates In Vitro Reconstitution of the C-S Bond-Forming P450 in Griseoviridin Biosynthesis.

Griseoviridin is a group A streptogramin natural product from Streptomyces with broad-spectrum antibacterial activity. A hybrid polyketide-nonribosomal peptide, it comprises a 23-membered macrocycle, an embedded oxazole motif, and a macrolactone with a unique ene-thiol linkage. Recent analysis of the griseoviridin biosynthetic gene cluster implicated SgvP, a cytochrome P450 monooxygenase, in late-stage installation of the critical C-S bond. While genetic and crystallographic experiments provided indirect evidence to support this hypothesis, the exact function of SgvP has never been confirmed biochemically. Herein, we report a convergent total synthesis of pre-griseoviridin, the putative substrate of P450 SgvP and precursor to griseoviridin. Our strategy features concise and rapid assembly of two fragments joined via sequential peptide coupling and Stille macrocyclization. Access to pre-griseoviridin then enabled in vitro validation of SgvP as the C-S bond-forming P450 during griseoviridin biosynthesis, culminating in a nine-step chemoenzymatic synthesis of griseoviridin.

Functional studies of McSTE24, McCYP305a1, and McJHEH, three essential genes act in cantharidin biosynthesis in the blister beetle (Coleoptera: Meloidae).

Cantharidin is a toxic defensive substance secreted by most blister beetles when attacked. It has been used to treat many complex diseases since ancient times and has recently regained popularity as an anticancer agent. However, the detailed mechanism of the cantharidin biosynthesis has not been completely addressed. In this study, we cloned McSTE24 (encoding STE24 endopeptidase) from terpenoid backbone pathway, McCYP305a1 (encoding cytochrome P450, family 305) and McJHEH [encoding subfamily A, polypeptide 1 and juvenile hormone (JH) epoxide hydrolase] associated to JH synthesis/degradation in the blister beetle Mylabris cichorii (Linnaeus, 1758, Coleoptera: Meloidae). Expression pattern analyses across developmental stages in adult males revealed that the expressions of 3 transcripts were closely linked to cantharidin titer exclusively during the peak period of cantharidin synthesis (20-25 days old). In contrast, at other stages, these genes may primarily regulate different biological processes. When RNA interference with double-stranded RNA suppressed the expressions of the 3 genes individually, significant reductions in cantharidin production were observed in males and also in females following McJHEH knockdown, indicating that these 3 genes might primarily contribute to cantharidin biosynthesis in males, but not in females, while females could self-synthesis a small amount of cantharidin. These findings support the previously hypothesized sexual dimorphism in cantharidin biosynthesis during the adult phase. McCYP305a1 collaborates with its upstream gene McSTE24 in cantharidin biosynthesis, while McJHEH independently regulates cantharidin biosynthesis in males.

Cyprocide selectively kills nematodes via cytochrome P450 bioactivation.

Left unchecked, plant-parasitic nematodes have the potential to devastate crops globally. Highly effective but non-selective nematicides are justifiably being phased-out, leaving farmers with limited options for managing nematode infestation. Here, we report our discovery of a 1,3,4-oxadiazole thioether scaffold called Cyprocide that selectively kills nematodes including diverse species of plant-parasitic nematodes. Cyprocide is bioactivated into a lethal reactive electrophilic metabolite by specific nematode cytochrome P450 enzymes. Cyprocide fails to kill organisms beyond nematodes, suggesting that the targeted lethality of this pro-nematicide derives from P450 substrate selectivity. Our findings demonstrate that Cyprocide is a selective nematicidal scaffold with broad-spectrum activity that holds the potential to help safeguard our global food supply.

Investigation of cannabidiol-induced cytotoxicity in human hepatic cells.

Cannabidiol (CBD) is one of the primary cannabinoids present in extracts of the plant Cannabis sativa L. A CBD-based drug, Epidiolex, has been approved by the U.S. FDA for the treatment of seizures in childhood-onset epileptic disorders. Although CBD-associated liver toxicity has been reported in clinical studies, the underlying mechanisms remain unclear. In this study, we demonstrated that CBD causes cytotoxicity in primary human hepatocytes and hepatic HepG2 cells. A 24-h CBD treatment induced cell cycle disturbances, cellular apoptosis, and endoplasmic reticulum (ER) stress in HepG2 cells. A potent ER stress inhibitor, 4-phenylbutyrate, markedly attenuated CBD-induced apoptosis and cell death. Additionally, we investigated the role of cytochrome P450 (CYP)-mediated metabolism in CBD-induced cytotoxicity using HepG2 cell lines engineered to express 14 individual CYPs. We identified CYP2C9, 2C19, 2D6, 2C18, and 3A5 as participants in CBD metabolism. Notably, cells overexpressing CYP2C9, 2C19, and 2C18 produced 7-hydroxy-CBD, while cells overexpressing CYP2C9, 2C19, 2D6, and 2C18 generated 7-carboxy-CBD. Furthermore, CBD-induced cytotoxicity was significantly attenuated in the cells expressing CYP2D6. Taken together, these data suggest that cell cycle disturbances, apoptosis, and ER stress are associated with CBD-induced cytotoxicity, and CYP2D6-mediated metabolism plays a critical role in decreasing the cytotoxicity of CBD.

Mechanism of azole resistance in Candida vulturna, an emerging multidrug resistant pathogen related with Candida haeumulonii and Candida auris.

BACKGROUND: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. OBJECTIVES: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. METHODS: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. RESULTS: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. CONCLUSIONS: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus.

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

A Malpighian Tubule-Specific P450 Gene SlCYP9A75a Contributes to Xenobiotic Tolerance in Spodoptera litura.

Phytophagous insects are more predisposed to evolve insecticide resistance than other insect species due to the "preadaptation hypothesis". Cytochrome P450 monooxygenases have been strongly implicated in insecticide and phytochemical detoxification in insects. In this study, RNA-seq results reveal that P450s of Spodoptera litura, especially the CYP3 clan, are dominant in cyantraniliprole, nicotine, and gossypol detoxification. The expression of a Malpighian tubule-specific P450 gene, SlCYP9A75a, is significantly upregulated in xenobiotic treatments except α-cypermethrin. The gain-of-function and loss-of-function analyses indicate that SlCYP9A75a contributes to cyantraniliprole, nicotine, and α-cypermethrin tolerance, and SlCYP9A75a is capable of binding to these xenobiotics. This study indicates the roles of inducible SlCYP9A75a in detoxifying man-made insecticides and phytochemicals and may provide an insight into the development of cross-tolerance in omnivorous insects.

Chemoenzymatic Synthesis of Fluorinated Mycocyclosin Enabled by the Engineered Cytochrome P450-Catalyzed Biaryl Coupling Reaction.

Installing fluorine atoms onto natural products holds great promise for the generation of fluorinated molecules with improved or novel pharmacological properties. The enzymatic oxidative carbon-carbon coupling reaction represents a straightforward strategy for synthesizing biaryl architectures, but the exploration of this method for producing fluorine-substituted derivatives of natural products remains elusive. Here, in this study, we report the protein engineering of cytochrome P450 from Mycobacterium tuberculosis (MtCYP121) for the construction of a series of new-to-nature fluorine-substituted Mycocyclosin derivatives. This protocol takes advantage of a "hybrid" chemoenzymatic procedure consisting of tyrosine phenol lyase-catalyzed fluorotyrosine preparation from commercially available fluorophenols, intermolecular chemical condensation to give cyclodityrosines, and an engineered MtCYP121-catalyzed intramolecular biphenol coupling reaction to complete the strained macrocyclic structure. Computational mechanistic studies reveal that MtCYP121 employs Cpd I to abstract a hydrogen atom from the proximal phenolic hydroxyl group of the substrate to trigger the reaction. Then, conformational change makes the two phenolic hydroxyl groups close enough to undergo intramolecular hydrogen atom transfer with the assistance of a pocket water molecule. The final diradical coupling process completes the intramolecular C-C bond formation. The efficiency of the biaryl coupling reaction was found to be influenced by various fluorine substitutions, primarily due to the presence of distinct binding conformations.

Detection of a Kinetically Competent Compound-I Intermediate in the Vancomycin Biosynthetic Enzyme OxyB.

Cytochrome P450 enzymes are abundantly encoded in microbial genomes. Their reactions have two general outcomes, one involving oxygen insertion via a canonical "oxygen rebound" mechanism and a second that diverts from this pathway and leads to a wide array of products, notably intramolecular oxidative cross-links. The antibiotic of-last-resort, vancomycin, contains three such cross-links, which are crucial for biological activity and are installed by the P450 enzymes OxyB, OxyA, and OxyC. The mechanisms of these enzymes have remained elusive in part because of the difficulty in spectroscopically capturing transient intermediates. Using stopped-flow UV/visible absorption and rapid freeze-quench electron paramagnetic resonance spectroscopies, we show that OxyB generates the highly reactive compound-I intermediate, which can react with a model vancomycin peptide substrate in a kinetically competent fashion to generate product. Our results have implications for the mechanism of OxyB and are in line with the notion that oxygen rebound and oxidative cross-links share early steps in their catalytic cycles.

Azole resistance in Aspergillus flavus and associated fitness cost.

BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.