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1.
Biochem Biophys Res Commun ; 711: 149914, 2024 Jun 04.
Article En | MEDLINE | ID: mdl-38608434

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.


Aedes , Aedes/enzymology , Aedes/metabolism , Animals , Glutathione Transferase/metabolism , Glutathione/metabolism , Ecdysone/metabolism , Insect Proteins/metabolism , Substrate Specificity , Steroid Isomerases/metabolism , Steroid Isomerases/genetics , Mosquito Vectors/metabolism , Ketosteroids/metabolism , Ketosteroids/chemistry
2.
Cancers (Basel) ; 16(4)2024 Feb 12.
Article En | MEDLINE | ID: mdl-38398153

Protein engineering can be used to tailor enzymes for medical purposes, including antibody-directed enzyme prodrug therapy (ADEPT), which can act as a tumor-targeted alternative to conventional chemotherapy for cancer. In ADEPT, the antibody serves as a vector, delivering a drug-activating enzyme selectively to the tumor site. Glutathione transferases (GSTs) are a family of naturally occurring detoxication enzymes, and the finding that some of them are overexpressed in tumors has been exploited to develop GST-activated prodrugs. The prodrug Telcyta is activated by GST P1-1, which is the GST most commonly elevated in cancer cells, implying that tumors overexpressing GST P1-1 should be particularly vulnerable to Telcyta. Promising antitumor activity has been noted in clinical trials, but the wildtype enzyme has modest activity with Telcyta, and further functional improvement would enhance its usefulness for ADEPT. We utilized protein engineering to construct human GST P1-1 gene variants in the search for enzymes with enhanced activity with Telcyta. The variant Y109H displayed a 2.9-fold higher enzyme activity compared to the wild-type GST P1-1. However, increased catalytic potency was accompanied by decreased thermal stability of the Y109H enzyme, losing 99% of its activity in 8 min at 50 °C. Thermal stability was restored by four additional mutations simultaneously introduced without loss of the enhanced activity with Telcyta. The mutation Q85R was identified as an important contributor to the regained thermostability. These results represent a first step towards a functional ADEPT application for Telcyta.

3.
Biomolecules ; 13(6)2023 06 11.
Article En | MEDLINE | ID: mdl-37371556

Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the orders Diptera and Lepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and the suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquito Anopheles gambiae is a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity of these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Aminoacid residue Asp111 in Nobo is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.


Anopheles , Malaria , Animals , Mosquito Vectors , Insecta , Steroids , Mammals , Ketosteroids
4.
Front Mol Biosci ; 8: 765970, 2021.
Article En | MEDLINE | ID: mdl-34881290

In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M-1s-1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3ß-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s-1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.

5.
Biochem Biophys Rep ; 27: 101078, 2021 Sep.
Article En | MEDLINE | ID: mdl-34286113

The common marmoset Callithrix jacchus encodes two glutathione transferase (GST) enzymes with ketosteroid double-bond isomerase activity. The most active enzyme is CjaGST A3-3 showing a specific activity with 5-androsten-3,17-dione (Δ5-AD) of 62.1 ± 1.8 µmol min-1 mg-1, and a kcat value of 261 ± 49 s-1. The second ketosteroid isomerase CjaGST A1-1 has a 30-fold lower specific activity with Δ5-AD and a 37-fold lower kcat value. Thus, the marmoset CjaGST A3-3 would be the main contributor to the biosynthesis of the steroid hormones testosterone and progesterone, like the human ortholog HsaGST A3-3. Two residues differ in the H-site of the 91.4% sequence identical CjaGST A1-1 and CjaGST A3-3, and modeling of the structures suggests that the bulky phenyl ring of Phe111 in CjaGST A1-1 causes steric hindrance in the binding of the steroid substrate. Tributyltin acetate (IC50=0.16 ± 0.004 µM) and ethacrynic acid (IC50=3.3 ± 0.2 µM) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.

6.
Int J Mol Sci ; 22(8)2021 Apr 15.
Article En | MEDLINE | ID: mdl-33920860

Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC50 value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.


Glutathione S-Transferase pi/metabolism , Veterinary Medicine , Amino Acid Sequence , Animals , Biocatalysis , Dogs , Glutathione S-Transferase pi/antagonists & inhibitors , Glutathione S-Transferase pi/chemistry , Glutathione S-Transferase pi/isolation & purification , Humans , Models, Molecular , Prodrugs/chemistry , Prodrugs/pharmacology , Protein Structure, Tertiary , Substrate Specificity
7.
Environ Pollut ; 268(Pt B): 115960, 2021 Jan 01.
Article En | MEDLINE | ID: mdl-33162212

Organotin compounds are highly toxic environmental pollutants with neurotoxic and endocrine-disrupting effects. They are potent inhibitors of glutathione transferases (GSTs), thus impeding their detoxication and antioxidant functions. Several GSTs, including equine GST A3-3 (EcaGST A3-3), exhibit steroid double-bond isomerase activity and are involved in the biosynthesis of testosterone and progesterone. We have performed enzyme kinetics analyses of the inhibition of EcaGST A3-3 by organotin compounds. We have also solved crystal structures of EcaGST A3-3 in complexes with glutathione, and with glutathione together with covalently bound triethyltin. Our structural data indicate that the tin atom forms strong bonds with a covalent character not only with the glutathione, but also with a tyrosyl residue of the enzyme itself, thereby preventing the release of the glutathione-organotin adduct and completely blocking the enzyme function. This work presents a structural basis for the general mechanism of GST inhibition by organotin compounds and contributes to the understanding of their neurotoxic and endocrine disrupting effects.


Environmental Pollutants , Organotin Compounds , Animals , Glutathione , Glutathione Transferase , Horses , Organotin Compounds/toxicity , Steroids
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