Accumulation of 4-Deoxy-7-Hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-type FgTri1 Gene

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain’s genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17β-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol.

Overexpression of a Cytochrome P450 Monooxygenase Involved in Orobanchol Biosynthesis Increases Susceptibility to Fusarium Head Blight

Fusarium Head Blight (FHB) is a cereal disease caused primarily by the ascomycete fungus Fusarium graminearum with public health issues due to the production of mycotoxins including deoxynivalenol (DON). Genetic resistance is an efficient protection means and numerous quantitative trait loci have been identified, some of them related to the production of resistance metabolites. In this study, we have functionally characterized the Brachypodium distachyon BdCYP711A29 gene encoding a cytochrome P450 monooxygenase (CYP). We showed that BdCYP711A29 belongs to an oligogenic family of five members. However, following infection by F. graminearum, BdCYP711A29 is the only copy strongly transcriptionally induced in a DON-dependent manner. The BdCYP711A29 protein is homologous to the Arabidopsis thaliana MAX1 and Oryza sativa MAX1-like CYPs representing key components of the strigolactone biosynthesis. We show that BdCYP711A29 is likely involved in orobanchol biosynthesis. Alteration of the BdCYP711A29 sequence or expression alone does not modify plant architecture, most likely because of functional redundancy with the other copies. B. distachyon lines overexpressing BdCYP711A29 exhibit an increased susceptibility to F. graminearum, although no significant changes in defense gene expression were detected. We demonstrate that both orobanchol and exudates of Bd711A29 overexpressing lines stimulate the germination of F. graminearum macroconidia. We therefore hypothesize that orobanchol is a susceptibility factor to FHB.

Structural basis for selective oxidation of phosphorylated ethylphenols by cytochrome P450 monooxygenase CreJ

Selective oxidation of C-H bonds in alkylphenols holds great significance for not only structural derivatization in pharma- and bio-manufacturing but also biological degradation of these toxic chemicals in environmental protection. A unique chemomimetic biocatalytic system using enzymes from a p-cresol biodegradation pathway has recently been developed. As the central biocatalyst, the cytochrome P450 monooxygenase CreJ oxidizes diverse p- and m-alkylphenyl phosphates with perfect stereoselectivity at different efficiencies. However, the mechanism of regio- and stereoselectivity of this chemomimetic biocatalytic system remained unclear. Here, using p- and m-ethylphenyl substrates, we elucidate the CreJ-catalyzed key steps for selective oxidations. The crystal structure of CreJ in complex with m-ethylphenyl phosphate was solved and compared with its complex structure with p-ethylphenyl phosphate isomer. The results indicate that the conformational changes of substrate-binding residues are slight, while the substrate promiscuity is achieved mainly by the available space in the catalytic cavity. Moreover, the catalytic preferences of regio- and stereoselective hydroxylation for the two ethylphenyl substrates is explored by molecular dynamics simulations. The ethyl groups in the complexes display different flexibility, and the distances of the active oxygen to Hpro-S and Hpro-R of methylene agree with the experimental stereoselectivity. The regioselectivity can be explained by the distances and bond dissociation energy. These results provide not only the mechanistic insights of CreJ into its regio- and stereoselectivity but also the structural basis for further P450 enzyme design and engineering. Importance The key cytochrome P450 monooxygenase CreJ showed excellent regio- and stereoselectivity in the oxidation of various alkylphenol substrates. C-H bond functionalization of these toxic alkylphenols holds great significance both for biological degradation of these environmental chemicals and production of value-added structural derivatives in pharmaceutical and biochemical industries. Our results, combined with in vitro enzymatic assays, crystal structure determination of enzyme-substrate complex, and molecular dynamics simulations, provide not only significant mechanism elucidation of the regio- and stereoselective catalyzation mediated by CreJ, but also the promising directions for the future engineering efforts of this enzyme towards more useful products. It also has great extendable potentials to couple this multifunctional P450 biocatalyst with other post modifying enzymes (e.g. hydroxyl based glycosylase) to access more alkylphenol derived high-value chemicals through environment-friendly biocatalysis and biotransformation.

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine

Engineered cytochrome P450 monooxygenase CYP154E1 enables the effective synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine via N-demethylation and regio- and stereoselective hydroxylation of (R)-ketamine.

Cytochrome P450 monooxygenase of Acanthamoeba castellanii participates in resistance to polyhexamethylene biguanide treatment

Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK). Polyhexamethylene biguanide (PHMB) is a topical application for AK treatment. However, PHMB is not entirely effective against all Acanthamoeba strains or isolates. The mechanisms by which Acanthamoeba protects itself against extreme drug conditions without encystation are still unknown. According to a previous study, cytochrome P450 monooxygenase (CYP450MO) plays an important role in the oxidative biotransformation of numerous drugs related to metabolism. In this study, a CYP450MO fragment was inserted into the pGAPDH-EGFP vector and transfected into Acanthamoeba castellanii. We found that CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. Moreover, we also found that encystation-related genes such as cellulose synthase I (CSI), encystation-mediating serine proteinase (EMSP), and autophagy-related protein 8 (ATG8) expression levels were not significantly different between Acanthamoeba transfected by pGAPDH-EGFP or pGAPDH-EGFP-CYP450MO. We suggest that Acanthamoeba transfected by pGAPDH-EGFP-CYP450MO may not induce encystation-related genes to resist PHMB treatment. In conclusion, these findings indicate that CYP450MO may be an additional target when PHMB is used for treatment of amoebic keratitis.

Structure of the fungal hydroxylase, CYP505A30, and rational transfer of mutation data from CYP102A1 to alter regioselectivity

CYP505A30 is a fungal, self-sufficient cytochrome P450 monooxygenase that can selectively oxyfunctionalise n-alkanes, fatty alcohols, and fatty acids. From alkanes, it produces a mixture of non-vicinal diols by two sequential...