Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance

The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (K_cat = 3.2 pmol/min/pmol P450, K_m = 36 μM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations.     

Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control inAfrica. Elucidation of underlying resistance mechanisms is crucial to improve the successof future control programs. A positional cloning approach was used to identify genesconferring resistance in the uncharacterised rp2 quantitative trait locus (QTL)previously detected in this vector using F6 advanced intercross lines (AIL). A113 kb BAC clone spanning rp2 was identified and sequenced revealing acluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary toA. gambiae, AfCYP6M1 is triplicated in A. funestus, whileAgCYP6Z2 orthologue is absent. Five hundred and sixty-five new singlenucleotide polymorphisms (SNPs) were identified for genetic mapping from rp2P450s and other genes revealing high genetic polymorphisms with one SNP every36 bp. A significant genotype/phenotype association was detected forrp2 P450s but not for a cluster of cuticular protein genes previouslyassociated with resistance in A. gambiae. QTL mapping using F6 AIL confirms therp2 QTL with an increase logarithm of odds score of 5. Multiplex geneexpression profiling of 15 P450s and other genes around rp2 followed byindividual validation using qRT–PCR indicated a significant overexpression in theresistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3,AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective foldchange of 11.2, 6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 andAfCYP6Z3 identified amino acid changes potentially associated with resistancefurther indicating that these genes are controlling the pyrethroid resistance explained bythe rp2 QTL. The characterisation of this rp2 QTL significantly improvesour understanding of resistance mechanisms in A. funestus.     

The production of recombinant cationic α‐helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum

Synthetic linear antimicrobial peptides with cationic α‐helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C‐terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein.     

Stimulation of the HIV‐1 integrase enzymatic activity and cDNA integration by a peptide derived from the integrase protein

Here we describe the features of a peptide that was selected from the human immunodeficiency virus Type 1 (HIV‐1) Integrase (IN) peptide library which interacts with both, the viral Rev and IN proteins. Because of its ability to stimulate the IN enzymatic activity this peptide was designated INS (IN stimulatory). Modification of its amino acid sequence revealed that replacement of its C‐terminal lysine by glutamic acid (INS K188E) converts it from a stimulatory peptide to an inhibitory one. Both peptides promoted the dissociation of a previously described complex formed between Rev and IN whose formation results in IN inactivation. INS and INS K188E penetrated HIV‐1‐infected cells and caused stimulation and inhibition of viral genome integration, respectively. Using cultured cells infected with a ΔRev HIV revealed that INS can directly activate the viral IN. These results suggest that the stimulatory effect of INS in wild‐type virus‐infected cells is due to a dual effect: it dissociates the inactive Rev‐IN complex and directly activates the free IN. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 740–751, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Metabolism of Methoxychlor by the P450‐Monooxygenase CYP6G1 Involved in Insecticide Resistance of Drosophila melanogaster after Expression in Cell Cultures of Nicotiana tabacum

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1‐trichloro‐2,2‐bis(4‐methoxyphenyl)ethane, 1 ) against the background of endogenous enzymes of the corresponding non‐transgenic culture. The Cyp6g1‐transgenic cell culture metabolized 96% of applied methoxychlor (45.8 μg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 μg), whereas inhibition in the non‐transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid ( 6 ; 20 μg, 24 h) in the Cyp6g1‐transgenic culture by 82% in the presence of piperonyl butoxide (200 μg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor ( 1 ). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.     

Biosynthesis of the leucine derived α‐, β‐ and γ‐hydroxynitrile glucosides in barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) produces five leucine‐derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non‐cyanogenic HNGs are the β‐HNG epidermin and the γ‐HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP‐glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co‐expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi‐enzyme complex.     

Effect of different stock types and dietary protein levels on key enzyme activities of glycolysis‐gluconeogenesis,the pentose phosphate pathway and amino acid metabolism in Macrobrachium rosenbergii

A feeding trial was conducted to study the effect of dietary protein (DP) levels on the enzyme activities of different stocks of Macrobrachium rosenbergii juveniles, fed two diets prawn type diets either at optimum (ODP, 32%) or sub‐optimum (SDP, 27%) dietary protein. The activity of key enzymes: glycolysis, gluconeogenesis, amino acid catabolism, pentose phosphate pathway, and energy metabolism was examined on different stocks of M. rosenbergii juveniles in various tissues (muscle, hepatopancreas, and gill). Wild juveniles of M. rosenbergii were collected from the west coast of India, Gujarat (G), Maharashtra (M) and from the east coast of India, Andhra Pradesh (A), and raised in culture ponds of 200 m² at 1 juvenile m^‐2. The experiment was conducted for a period of 60 days. A Visible Implant Elastomer (VIE) Tag was injected into different prawn juveniles’ stocks A, (3.06–3.10 g), M, (0.80–1.01 g) and G (0.90–1.06 g) for the individual identification of each class. All the animals were acclimatized for 7 days before being released into the pond. Each of the two diets, the first with 27% SDP, termed the sub‐optimum level (S), and the second 32% DP, termed the optimum level (O), was fed in triplicate ponds. The weight gain of stocked prawn was significantly higher for G stock (3.03 ± 0.18 g) compared to M stock (2.53 ± 0.25 g) and A stock (1.33 ± 0.10 g). In addition weight gain in ODP was significantly higher (3.05 ± 0.22 g) compared to SDP (2.11 ± 0.17 g). Furthermore, both protein level and stock type had a significant (p ≤ 0.05) effect on the survival rate, specific growth rate, and feed conversion ratio of the prawn. A significant interaction was recorded between metabolic enzyme activities and the variation of dietary protein levels and differences in M. rosenbergii stocks, in which stock “G” and “M” exhibited healthier enzyme physiological status than stock “A”. The findings presented here may conclude that the enzymes quickly respond to dietary protein manipulations of cultured shrimp. The results can be useful in identifying the optimum stock (genotypes) in a given culture condition and subsequently higher production may be achieved.