scholarly journals A cytochrome P450 from juvenile mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis

2019 ◽  
Author(s):  
Nanxia Fu ◽  
Zhi-ling Yang ◽  
Yannick Pauchet ◽  
Christian Paetz ◽  
Wolfgang Brandt ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Fat Body ◽  
Mevalonate Pathway ◽  
Leaf Beetle ◽  
List Type ◽  
P450 Monooxygenase ◽  
Double Stranded Rna ◽  
Leaf Beetles ◽  
Phaedon Cochleariae

AbstractJuveniles of the leaf beetle Phaedon cochleariae synthesize iridoid via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to 8-hydroxygeraniol. Here we identify and characterize the geraniol 8-hydroxylase as a P450 monooxygenase using integrated transcriptomic and proteomic analyses. In the fat body, 73 individual cytochrome P450s were identified. The double stranded RNA (dsRNA)-mediated knock down of CYP6BH5 led to a significant reduction of 8-hydroxygeraniol-glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to 8-hydroxygeraniol. In addition to geraniol, CYP6BH5 also catalyzes other monoterpenols, such as nerol and citronellol, into the corresponding α, ω-dihydroxy compounds.HighlightsThe geraniol 8-hydroxylase in Phaedon cochleariae was identified as a cytochrome P450 CYP6BH5.RNA interference emphasized the importance of CYP6BH5 in iridoid biosynthesis.In vitro enzyme assays showed that recombinant CYP6BH5 is a substrate promiscuous enzyme, converting the ω-hydroxylation of geraniol, nerol, citronellol but not linalool.Homology modeling suggested the -OH group of the substrate plays an important role in coordinating the substrates with the enzyme’s catalytic cavity.

scholarly journals In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

2021 ◽  
Vol 22 (15) ◽  
pp. 7811
Author(s):  
Olufunmilayo Olukemi Akapo ◽  
Joanna M. Macnar ◽  
Justyna D. Kryś ◽  
Puleng Rosinah Syed ◽  
Khajamohiddin Syed ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Structural Analysis ◽  
In Silico ◽  
Web Application ◽  
Cytochrome P450 Monooxygenases ◽  
Agglomerative Clustering ◽  
Ligand Complex ◽  
P450 Monooxygenase ◽  
Study Results

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.

scholarly journals Reduction of Cellular Lipid Content by a Knockdown ofDrosophila PDP1γand Mammalian Hepatic Leukemia Factor

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Svetlana Dzitoyeva ◽  
Hari Manev
Keyword(s):  
Lipid Content ◽  
In Vitro Model ◽  
Fat Body ◽  
Double Stranded Rna ◽  
Mrna Content ◽  
Tissue Lipids ◽  
Vitro Model ◽  
Embryonic Factor ◽  
Site Binding

In exploring the utility of double-stranded RNA (dsRNA) injections for silencing thePAR-domain protein 1 (Pdp1)gene in adultDrosophila, we noticed a dramatic loss of fat tissue lipids. To verify that our RNAi approach produced the expectedPdp1knockdown, the abdominal fat tissues sections were stained with PDP1 antibodies. PDP1 protein immunostaining was absent in flies injected with dsRNA targeting a sequence common to all knownPdp1isoforms. Subsequent experiments revealed that lipid staining is reduced in flies injected with dsRNA against Pdp1γ(fat body specific) and not againstPdp1ε(predominantly involved in circadian mechanisms).DrosophilaPDP1γprotein shows a high homology to mammalian thyrotroph embryonic factor (TEF), albumin D site-binding protein (DBP), and hepatic leukemia factor (HLF) transcription factors. In an in vitro model of drug- (olanzapine-) induced adiposity in mouse 3T3-L1 cells, the mRNA content of HLF but not TEF and DBP was increased by the drug treatment. A knockdown of the HLF mRNA by transfecting the cultures with HLF dsRNA significantly reduced their lipid content. Furthermore, the HLF RNAi prevented olanzapine from increasing the cell lipid content. These results suggest that the PDP1/HLF system may play a role in physiological and drug-influenced lipid regulation.

The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone

2006 ◽  
Vol 34 (6) ◽  
pp. 1256-1260 ◽  
Author(s):  
K.F. Rewitz ◽  
R. Rybczynski ◽  
J.T. Warren ◽  
L.I. Gilbert
Keyword(s):  
Cytochrome P450 ◽  
Fat Body ◽  
Larval Instar ◽  
Primary Source ◽  
Pupal Stage ◽  
Prothoracic Gland ◽  
Cytochrome P450 Enzymes ◽  
Biosynthetic Activity ◽  
Prothoracic Glands

The developmental events occurring during moulting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the moulting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20E (20-hydroxyecdysone), are mediated by four cytochrome P450 (P450) enzymes, encoded by genes in the Halloween family. Orthologues of the Drosophila Halloween genes phantom (phm; CYP306A1), disembodied (dib; CYP302A1), shadow (sad; CYP315A1) and shade (shd; CYP314A1) were obtained from the endocrinological model insect, the tobacco hornworm Manduca sexta. Expression of these genes was studied and compared with changes in the ecdysteroid titre that controls transition from the larval to pupal stage. phm, dib and sad, which encode P450s that mediate the final hydroxylations in the biosynthesis of ecdysone, were selectively expressed in the prothoracic gland, the primary source of ecdysone during larval and pupal development. Changes in their expression correlate with the haemolymph ecdysteroid titre during the fifth (final) larval instar. Shd, the 20-hydroxylase, which converts ecdysone into the more active 20E, is expressed in tissues peripheral to the prothoracic glands during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The results indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses triggering moulting.

scholarly journals 3,6-Dichlorosalicylate Catabolism Is Initiated by the DsmABC Cytochrome P450 Monooxygenase System inRhizorhabdus dicambivoransNdbn-20

2017 ◽  
Vol 84 (4) ◽  
Author(s):  
Na Li ◽  
Li Yao ◽  
Qin He ◽  
Jiguo Qiu ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Bioinformatic Analysis ◽  
Heme Iron ◽  
Cytochrome P450 Monooxygenase ◽  
Monooxygenase System ◽  
Catabolic Pathway ◽  
P450 Monooxygenase ◽  
Content Type ◽  
Promising Target

ABSTRACTThe degradation of the herbicide dicamba is initiated by demethylation to form 3,6-dichlorosalicylate (3,6-DCSA) inRhizorhabdusdicambivoransNdbn-20. In the present study, a 3,6-DCSA degradation-deficient mutant, Ndbn-20m, was screened. A cluster,dsmR1DABCEFGR2, was lost in this mutant. The cluster consisted of nine genes, all of which were apparently induced by 3,6-DCSA. DsmA shared 30 to 36% identity with the monooxygenase components of reported three-component cytochrome P450 systems and formed a monophyletic branch in the phylogenetic tree. DsmB and DsmC were most closely related to the reported [2Fe-2S] ferredoxin and ferredoxin reductase, respectively. The disruption ofdsmAin strain Ndbn-20 resulted in inactive 3,6-DCSA degradation. WhendsmABC, but notdsmAalone, was introduced into mutant Ndbn-20m andSphingobium quisquiliarumDC-2 (which is unable to degrade salicylate and its derivatives), they acquired the ability to hydroxylate 3,6-DCSA. Single-crystal X-ray diffraction demonstrated that the DsmABC-catalyzed hydroxylation occurred at the C-5 position of 3,6-DCSA, generating 3,6-dichlorogentisate (3,6-DCGA). In addition, DsmD shared 51% identity with GtdA (a gentisate and 3,6-DCGA 1,2-dioxygenase) fromSphingomonassp. strain RW5. However, unlike GtdA, the purified DsmD catalyzed the cleavage of gentisate and 3-chlorogentisate but not 6-chlorogentisate or 3,6-DCGAin vitro. Based on the bioinformatic analysis and gene function studies, a possible catabolic pathway of dicamba inR. dicambivoransNdbn-20 was proposed.IMPORTANCEDicamba is widely used to control a variety of broadleaf weeds and is a promising target herbicide for the engineering of herbicide-resistant crops. The catabolism of dicamba has thus received increasing attention. Bacteria mineralize dicamba initially via demethylation, generating 3,6-dichlorosalicylate. However, the catabolism of 3,6-dichlorosalicylate remains unknown. In this study, we cloned a gene cluster,dsmR1DABCEFGR2, involved in 3,6-dichlorosalicylate degradation fromR. dicambivoransNdbn-20, demonstrated that the cytochrome P450 monooxygenase system DsmABC was responsible for the 5-hydroxylation of 3,6-dichlorosalicylate, and proposed a dicamba catabolic pathway. This study provides a basis to elucidate the catabolism of dicamba and has benefits for the ecotoxicological study of dicamba. Furthermore, the hydroxylation of salicylate has been previously reported to be catalyzed by single-component flavoprotein or three-component Rieske non-heme iron oxygenase, whereas DsmABC was the only cytochrome P450 monooxygenase system hydroxylating salicylate and its methyl- or chloro-substituted derivatives.

scholarly journals Co-Expression of a Homologous Cytochrome P450 Reductase Is Required for In Vivo Validation of the Tetranychus urticae CYP392A16-Based Abamectin Resistance in Drosophila

Insects ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 829
Author(s):  
Maria Riga ◽  
Aris Ilias ◽  
John Vontas ◽  
Vassilis Douris
Keyword(s):  
Cytochrome P450 ◽  
Tetranychus Urticae ◽  
Transcriptional Analysis ◽  
Pest Species ◽  
Cytochrome P450 Reductase ◽  
Transcription Activator ◽  
P450 Monooxygenase ◽  
P450 Reductase

Overexpression of the cytochrome P450 monooxygenase CYP392A16 has been previously associated with abamectin resistance using transcriptional analysis in the two-spotted spider mite Tetranychus urticae, an important pest species worldwide; however, this association has not been functionally validated in vivo despite the demonstrated ability of CYP392A16 to metabolize abamectin in vitro. We expressed CYP392A16 in vivo via a Gal4 transcription activator protein/Upstream Activating Sequence (GAL4/UAS) system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. We demonstrated that CYP392A16 expression confers statistically significant abamectin resistance in toxicity bioassays in Drosophila only when its homologous redox partner, cytochrome P450 reductase (TuCPR), is co-expressed in transgenic flies. Our study shows that the Drosophila model can be further improved, to facilitate the functional analysis of insecticide resistance mechanisms acting alone or in combination.

scholarly journals Does Ozone Alter the Attractiveness of Japanese White Birch Leaves to the Leaf Beetle Agelastica coerulea via Changes in Biogenic Volatile Organic Compounds (BVOCs): An Examination with the Y-Tube Test

Forests ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 58
Author(s):  
Noboru Masui ◽  
Tomoki Mochizuki ◽  
Akira Tani ◽  
Hideyuki Matsuura ◽  
Evgenios Agathokleous ◽  
...  
Keyword(s):  
Volatile Organic Compounds ◽  
Organic Compounds ◽  
Leaf Beetle ◽  
Ex Situ ◽  
Biogenic Volatile Organic Compounds ◽  
White Birch ◽  
Leaf Beetles ◽  
Elevated O3 ◽  
Volatile Organic

Elevated ground-level ozone (O3) reduced C-based defense chemicals; however, severe grazing damages were found in leaves grown in the low O3 condition of a free air O3-concentration enrichment (O3-FACE) system. To explain this phenomenon, this study investigates the role of BVOCs (biogenic volatile organic compounds) as signaling compounds for insect herbivores. BVOCs act as scents for herbivore insects to locate host plants, while some BVOCs show high reactivity to O3, inducing changes in the composition of BVOCs in atmospheres with elevated O3. To assess the aforementioned phenomenon, profiles of BVOCs emitted from birch (Betula platyphylla var. japonica Hara) leaves were analyzed ex situ, and Y-tube insect preference tests were conducted in vitro to study the insect olfactory response. The assays were conducted in June and August or September, according to the life cycle of the adult alder leaf beetle Agelastica coerulea Baly (Coleoptera: Chrysomelidae). The Y-tube tests revealed that the leaf beetles were attracted to BVOCs, and O3 per se had neither an attractant nor a repellent effect. BVOCs became less attractant when mixed with highly concentrated O3 (>80 ppb). About 20% of the total BVOCs emitted were highly O3-reactive compounds, such as β-ocimene. The results suggest that BVOCs emitted from the birch leaves can be altered by elevated O3, thus potentially reducing the attractiveness of leaves to herbivorous insects searching for food.

scholarly journals Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities

2006 ◽  
Vol 6 (3) ◽  
pp. 514-520 ◽  
Author(s):  
Francisco Ferrer-Sevillano ◽  
José M. Fernández-Cañón
Keyword(s):  
Cytochrome P450 ◽  
Aspergillus Nidulans ◽  
High Performance ◽  
Wild Type ◽  
Catabolic Pathway ◽  
Cdna Subtraction ◽  
P450 Monooxygenase ◽  
Mutant Strains ◽  
Low Efficiency

ABSTRACT Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

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