Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters k inact and K I were found to be 0.020 ± 0.001 min−1 and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs.

Phoslactomycins (PLMs) are a group of natural products belonging to a polyketides class.These polyketides are synthesized by sequential reaction catalyzed by a collection of enzymes activities called polyketide synthases. A polyketide is a large class of diverse compounds that are characterized by more than two carbonyl groups connected by single intervening carbon atoms. In other words, a polyketide is a polymer whose monomer is a ketide. The PLMs are also known as phosphazomycins or phospholines. These compounds were isolated based on antifungal and antitumor activities. This array of promising biological activities has stimulated research into the field of PLMs for treatment of various diseases such as aspergillosis. A significant success has been reported in understanding and manipulating PLM biosynthesis. However, its post-polyketide biosynthetic mechanism remains to be elucidated. In this study, we established steps needed to pave the way for the elucidation of the post-polyketide synthase tailoring steps in the phoslactomycin biosynthetic pathway. Various, biological activities of polyketide natural products are often linked with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polyketide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activity.In this study, the enzymes and genes responsible for the modification of the phoslactomycin moiety have been investigated to verify their functions and to study how ii they are coordinated to achieve the desired phoslactomycin. The proposed modification steps in the PLM biosynthesis pathway involves, PlmT4 a cytochrome P450 monooxygenase, PlmT5, a kinase, and PlmT8 an oxidoreductase. These enzymes were successfully cloned, overexpressed, and purified from an overexpression vector. Mutant strains for two genes plmT4 and plmT8 were either constructed or studied. The function of PlmT4 tailoring enzyme was characterized, by gene disruption and an in vitro enzyme activity assay. The isolation of PLM 1 an intermediate analog from plmT4 mutant strain and the observation of a malonylated PLMs, suggests that the malonyl side chain is introduced during polyketide chain formation These results, will pave the way to delineate the intermediary steps between the PLM PKS product(s) that is released from the PLM PKS and the formation of the final phoslactomycin.