Cytochrome P450-mediated detoxification is one of the most important mechanisms involved in insecticide resistance. However, the molecular basis of this mechanism and the physiological functions of P450s associated with insecticide resistance remain largely unknown. Here, we exploited the functional genomics and reverse genetic approaches to identify and characterize a P450 gene responsible for the majority of deltamethrin resistance observed in the QTC279 strain of Tribolium castaneum. We used recently completed whole-genome sequence of T. castaneum to prepare custom microarrays and identified a P450 gene, CYP6BQ9, which showed more than a 200-fold higher expression in the deltamethrin-resistant QTC279 strain when compared with its expression in the deltamethrin-susceptible Lab-S strain. Functional studies using both double-strand RNA (dsRNA)-mediated knockdown in the expression of CYP6BQ9 and transgenic expression of CYP6BQ9 in Drosophila melanogaster showed that CYP6BQ9 confers deltamethrin resistance. Furthermore, CYP6BQ9 enzyme expressed in baculovirus metabolizes deltamethrin to 4-hydroxy deltamethrin. Strikingly, we also found that unlike many P450 genes involved in insecticide resistance that were reported previously, CYP6BQ9 is predominantly expressed in the brain, a part of the central nervous system (CNS) containing voltage-gated sodium channels targeted by deltamethrin. Taken together, the current studies on the brain-specific insect P450 involved in deltamethrin resistance shed new light on the understanding of the molecular basis and evolution of insecticide resistance.functional genomics | insecticide resistance | RNA interference | transgenic expression | insecticide metabolism

OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg = 4-hydroxyphenylglycine, and Dpg = 3,5-dihydroxyphenylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB with a kcat of 0.1 s-1 and Km in the range 4-13 muM. Equilibrium binding of this substrate to OxyB, monitored by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a Kd of 17 +/- 5 muM.

During the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. An oxygenase (OxyB) from the vancomycin producer catalyzes the first of these coupling reactions to a significant extent only when the putative hexapeptide substrate is linked as a thioester to a peptide carrier domain (PCD) derived from the non‐ribosomal peptide synthetase (see picture).

[reaction: see text] Alkoxyamines A, which are readily prepared from commercially available starting materials, undergo efficient thermal radical carboaminoxylations onto various nonactivated alkenes to provide 1,4-functionalized malonates B in good to excellent yields. The experiments are very easy to conduct. The carboaminoxylations can be combined with radical cyclization and fragmentation processes.

Teil der Biosynthese von Glycopeptidantibiotika der Vancomycin‐Familie sind mehrere oxidative Phenolkupplungsreaktionen. Eine Oxygenase (OxyB) des Vancomycin‐Produzenten katalysiert die erste dieser Kupplungen nur dann merklich, wenn das vermutliche Hexapeptidsubstrat als Thioester an eine Peptidträgerdomäne (PCD) geknüpft ist, die sich von der nichtribosomalen Peptid‐Synthetase ableitet (siehe Bild).

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OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide-PCP conjugate (R-Leu(1)-R-Tyr(2)-S-Asn(3)-R-Hpg(4)-R-Hpg(5)-S-Tyr(6)-S-PCP) (Hpg = 4-hydroxyphenylglycine) into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work, the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides related in sequence to this model hexapeptide-PCP conjugate. Deletion of one or three residues from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri- (Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol coupling to occur. The related tetrapeptide-PCP thioester was not cyclized by OxyB, neither was a related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB.

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