RESUMEN
Melanin protects dark-pigmented fungi from environmental stresses and serves as an important virulence factor in plant and human pathogenic fungi. The enzymes of melanin biosynthesis thus represent interesting targets for the development of fungicides and new selective antimycotics. In Curvularia lunata, a facultative plant and human pathogen, melanin is produced from 1,8-dihydroxynaphthalene via the pentaketide pathway. Recently, the melanin biosynthetic enzyme trihydroxynaphthalene reductase (3HNR) of C. lunata was cloned and expressed in Escherichia coli, enabling further inhibition studies. Here, we have examined structurally different flavonoids (flavones, flavonols, isoflavones and flavanones) as inhibitors of recombinant 3HNR by following the NADP(+)-dependant oxidation of a non-physiological substrate, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one. At 40 microM substrate concentration the most potent inhibitors were five flavones that are hydroxylated at positions 5 and 7: apigenin (IC(50), 3.1 microM), acacetin (IC(50), 4.9 microM), diosmetin (IC(50), 5.7 microM), 5,7-dihydroxyflavone (IC(50), 5.8 microM) and luteolin (IC(50), 6.8 microM). Flavonol (kaempferol; IC(50), 7.9 microM), isoflavone (genistein; IC(50), >50 microM) and flavanone (naringenin; IC(50), 26 microM) derivates were less potent than their corresponding flavone analogue apigenin. Among the isoflavones and flavanones, biochanin A was the most active (IC(50), 12 microM). Kinetic studies confirmed that apigenin and biochanin A, the best inhibitors among the flavones and isoflavones, act as competitive inhibitors of 3HNR, with K(i) values of 1.2 microM and 6.5 microM, respectively. Docking of apigenin and biochanin A into the active site of C. lunata 3HNR revealed their possible binding modes, in which they are stacked between the phenol ring of Tyr208 and the coenzyme nicotinamide moiety, forming two H-bonds with Ser149 and Ser228, and Ser149 and Tyr163, respectively. In vivo inhibition study showed that apigenin and one of the less potent inhibitors, baicalein affect fungal pigmentation and growth. Knowing that the flavonoids are formed in plants in response to fungal attack, they can be considered as potential physiological inhibitors of 3HNR.
Asunto(s)
Ascomicetos/enzimología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Cinética , Melaninas/antagonistas & inhibidores , Melaninas/biosíntesis , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
Human hydroxysteroid dehydrogenase (HSD) AKR1C1 is a member of the aldo-keto reductase superfamily, and it functions mainly as a 20alpha-HSD. It catalyzes the reduction of the potent progesterone to the weak 20alpha-hydroxyprogesterone, and of 3alpha,5alpha-tetrahydroprogesterone (5alpha-THP; allopregnanolone) to 5alpha-pregnane-3alpha,20alpha-diol. AKR1C1 thus decreases the levels of progesterone and 5alpha-THP in peripheral tissue. Progesterone inhibits cell proliferation, stimulates differentiation of endometrial cells, and is also important for maintenance of pregnancy, while 5alpha-THP allosterically modulates the activity of the gamma-aminobutyric acid receptor. Inhibitors of AKR1C1 are thus potential agents for treatment of endometrial cancer and endometriosis, as well as other diseases like premenstrual syndrome, catamenial epilepsy and depressive disorders.We have synthesized a series of pyrimidine, phthalimido and athranilic acid derivatives, and have here examined their inhibitory properties towards AKR1C1. A common aldo-keto reductase substrate, 1-acenaphthenol, was used to monitor the NAD(+)-dependent oxidation catalyzed by AKR1C1. The most potent inhibitors of AKR1C1 were the pyrimidine derivative N-benzyl-2-(2-(4-methoxybenzyl)-6-oxo-1,6-dihydropyrimidin-4-yl)acetamide (K(i)=17 microM) and the anthranilic acid derivative 2-(((2',3-dichlorobiphenyl-4-yl)carbonyl)(methyl)amino)benzoic acid (K(i)=33 microM), both of which are non-competitive inhibitors.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ftalimidas/farmacología , Pirimidinas/farmacología , ortoaminobenzoatos/farmacología , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Pirimidinas/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , ortoaminobenzoatos/químicaRESUMEN
Four soluble human 3 alpha-hydroxysteroid dehydrogenase (HSD) isoforms exist which are aldo-keto reductase (AKR) superfamily members. They share 86% sequence identity and correspond to: AKR1C1 (20 alpha(3 alpha)-HSD); AKR1C2 (type 3 3 alpha-HSD and bile-acid binding protein); AKR1C3 (type 2 3 alpha-HSD and type 5 17 beta-HSD); and AKR1C4 (type 1 3 alpha-HSD). Each of the homogeneous recombinant enzymes are plastic and display 3-, 17- and 20-ketosteroid reductase and 3 alpha- 17 beta- and 20 alpha-hydroxysteroid oxidase activities with different k(cat)/K(m) ratios in vitro. The crystal structure of the AKR1C2.NADP(+).ursodeoxycholate complex provides an explanation for this functional plasticity. Ursodeoxycholate is bound backwards (D-ring in the A-ring position) and upside down (beta-face of steroid inverted) relative to the position of 3-ketosteroids in the related rat liver 3 alpha-HSD (AKR1C9) structure. Transient transfection indicates that in COS-1 cells, AKR1C enzymes function as ketosteroid reductases due to potent inhibition of their oxidase activity by NADPH. By acting as ketosteroid reductases they may regulate the occupancy of the androgen, estrogen and progesterone receptors. RT-PCR showed that AKRs are discretely localized. AKR1C4 is virtually liver specific, while AKR1C2 and AKR1C3 are dominantly expressed in prostate and mammary gland. AKR1C genes are highly conserved in structure and may be transcriptionally regulated by steroid hormones and stress.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Exones , Humanos , Intrones , Isoenzimas/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Ascomicetos/enzimología , 17-Hidroxiesteroide Deshidrogenasas/genética , Ascomicetos/genética , Humanos , Modelos Moleculares , Filogenia , Conformación Proteica , Especificidad por SustratoRESUMEN
17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) has recently been characterized. Since its function is still unclear, we performed substrate specificity studies to obtain some indications about its physiological function. Different steroids were studied as putative substrates of recombinant 17beta-HSDcl, androgens and estrogens, brassinosteroids, and the fungal steroid herbarulid. Among these androgens and estrogens were most efficiently converted. The following substrates in decreasing order were best reduced: 4-estrene-3,17-dione, 5alpha-androstane-3,17-dione, 4-androstene-3,17-dione and estrone. Two typical inhibitors were tested: carbenoxolone--a representative inhibitor of the SDR family and quercetin--a diagnostic inhibitor of carbonyl reductases. Among these two quercetin was more efficient. Expression studies revealed that 17beta-HSDcl is mainly expressed in the stationary phase of growth indicating its possible involvement in secondary metabolism.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Ascomicetos/enzimología , Etiocolanolona/análogos & derivados , Proteínas Fúngicas/metabolismo , Expresión Génica , 17-Hidroxiesteroide Deshidrogenasas/análisis , 17-Hidroxiesteroide Deshidrogenasas/genética , Andrógenos/metabolismo , Androstenodiona/metabolismo , Carbenoxolona/farmacología , Inhibidores Enzimáticos/farmacología , Estrógenos/metabolismo , Estrona/metabolismo , Etiocolanolona/metabolismo , Quercetina/farmacología , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
17beta-Hydroxysteroid dehydrogenase (17beta-HSD) from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) catalyses the reduction of steroids and of several o- and p-quinones. After purification of the enzyme, its partial amino acid sequence was determined. A PCR fragment amplified with primers derived from peptide sequences was generated for screening the Coch. lunatus cDNA library. Three independent full-length cDNA clones were isolated and sequenced, revealing an 810-bp open reading frame encoding a 270-amino-acid protein. After expression in Escherichia coli and purification to homogeneity, the enzyme was found to be active towards androstenedione and menadione, and was able to form dimers of Mr 60000. The amino acid sequence of the novel 17beta-HSD demonstrated high homology with fungal carbonyl reductases, such as versicolorin reductase from Emericella nidulans (Aspergillus nidulans; VerA) and Asp. parasiticus (Ver1), polyhydroxynaphthalene reductase from Magnaporthe grisea, the product of the Brn1 gene from Coch. heterostrophus and a reductase from Colletotrichum lagenarium, which are all members of the short-chain dehydrogenase/reductase superfamily. 17beta-HSDcl is the first discovered fungal 17beta-hydroxysteroid dehydrogenase belonging to this family. The primary structure of this enzyme may therefore help to elucidate the evolutionary history of steroid dehydrogenases.