Expression of 17beta-hydroxysteroid dehydrogenases in mesophilic and extremophilic yeast

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes responsible for reversible interconversions of biologically active 17-hydroxy and inactive 17-keto steroids. We have performed a survey of 17beta-HSD activity in yeast. Constitutive 17beta-HSD activity was found in three mesophilic yeast species: Candida tropicalis, Cryptococcus tsukubaensis, and Saccharomyces cerevisiae as well as in three extremophilic black yeast species: Hortaea werneckii, Trimmatostroma salinum, and Phaeotheca triangularis, indicating that 17beta-HSD activity is widely distributed among yeast. In extremophilic black yeast, NaCl modulated enzyme activity. Enzymes resembling 17beta-HSD from the filamentous fungus Cochliobolus lunatus were detected in Trimmatostroma salinum and Phaeotheca triangularis. Sequences with identity to the Saccharomyces cerevisiae YBR159w gene were not observed in other yeast species possessing a similar enzyme activity. The results suggest the existence of at least three different types of 17beta-HSD in yeast.     

Membrane-bound human 3beta-hydroxysteroid dehydrogenase: overexpression with His-tag using a baculovirus system and single-step purification

The membrane-bound human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni(2+)-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C(12)E(8). High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K(m) of 1.7 microM and a V(max) of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme.     

Spironolactone-related inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities.

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.     

17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: structural and functional aspects

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.     

Temperature affects the production,activity and stability of ligninolytic enzymes in<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and<Emphasis Type="Italic">Trametes versicolor</Emphasis>

Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with cellulose as a sole C source and high C/N ratio. The fungi were able to grow and produce laccase and Mn-peroxidase (MnP) at 5-35 degrees C, the highest production being recorded at 25-30 degrees C in P. ostreatus and at 35 degrees C in T. versicolor. Production of both enzymes at 10 degrees C accounted only for 4-20% of the maximum value. Temperature optima for enzyme activity were 50 and 55 degrees C for P. ostreatus and T. versicolor laccases, respectively, and 60 degrees C for MnP. Temperatures causing 50% loss of activity after 24 h were 32 and 47 degrees C for laccases and 36 and 30 degrees C for MnP from P. ostreatus and T. versicolor, respectively.     

Production of lignocellulose-degrading enzymes and changes in soil bacterial communities during the growth ofPleurotus ostreatus in soil with different carbon content

The extracellular enzyme activity and changes in soil bacterial community during the growth of the ligninolytic fungus Pleurotus ostreatus were determined in nonsterile soil with low and high available carbon content. In soil with P. ostreatus, the activity of ligninolytic enzymes laccase and Mn-peroxidase was several orders of magnitude higher than in soil without the fungus. Addition of lignocellulose to soil increased the activity of cellulolytic fungi and the production of Mn-peroxidase by P. ostreatus. The counts of heterotrophic bacteria were more significantly affected by the presence of lignocellulose than by P. ostreatus. The effects of both substrate addition and time (succession) were more significant factors affecting the soil bacterial community than the presence of P. ostreatus. Bacterial community structure was affected by fungal colonization in low carbon soil, where a decrease of diversity and changes in substrate utilization profiles were detected.     

Expression of aromatase and 17beta-hydroxysteroid dehydrogenase types 1, 7 and 12 in breast cancer. An immunocytochemical study

It is known that there is a local biosynthesis of estradiol (E2) in breast carcinoma. The steroidogenic enzymes involved in E2 formation are aromatase which transforms testosterone into E2 and androstenedione into estrone (E1) and reductive 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) which convert E1 into E2. Using immunocytochemistry, we have studied the expression of aromatase and the three reductive 17beta-HSDs 17beta-HSD types 1, 7 and 12 in 41 specimens of female human breast carcinoma and adjacent non-malignant tissues. These results were correlated with the estrogen receptor alpha (ERalpha) and beta (ERbeta), progesterone receptor, androgen receptor, CDC47 and c-erb B-2 expressions and with the tumor stages. Aromatase was found in 58%, 17beta-HSD type 7 in 47% and 17beta-HSD type 12 in 83% of the breast cancer specimens. The 17beta-HSD type 1 could be detected in only one tumor. A significant correlation was observed between the aromatase, 17beta-HSD type 7 and 17beta-HSD type 12 expression, as well as between each of the two enzymes 17beta-types 7 and 12 and the ERbeta expression. The expression of 17beta-HSD type 12 was significantly higher in breast carcinoma specimens than in normal tissue. There was also a significant association of CDC 47 expression with ERbeta, AR and 17beta-HSD type 12. The results indicate that aromatase, 17beta-HSD type 7 and 17beta-HSD type 12, but not 17beta-HSD type 1, are commonly expressed in human breast cancer. Moreover, the high expression of both 17beta-HSD type 12 and ERbeta in breast carcinoma cells may play a role in the development and/or progression of breast cancer.     

Chemical synthesis of 16beta-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17beta-hydroxysteroid dehydrogenase and binding affinity on steroid receptors

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.     

New inhibitors of fungal 17beta-hydroxysteroid dehydrogenase based on the [1,5]-benzodiazepine scaffold

The synthesis and activity of a new series of non-steroidal inhibitors of 17beta-hydroxysteroid dehydrogenase that are based on a 1,5-benzodiazepine scaffold are presented. Their inhibitory potential was screened against 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. Some of these compounds are potent inhibitors of 17beta-HSDcl activity, with IC50 values in the low micromolar range and represent promising lead compounds that should be further developed and investigated as inhibitors of human 17beta-HSD isoforms, which are the enzymes associated with the development of many hormone-dependent and neuronal diseases.     

Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus

Nineteen fungi were tested for their ability to degrade aflatoxin B1 (AFB1). An extracellular enzyme from the edible mushroom Pleurotus ostreatus showed afaltoxin-degradation activity detected by thin-layer chromatography (TLC). An enzyme with this activity was purified by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. Optimum activities were found in the pH range between 4.0 and 5.0 and at 25 degrees C. Also, degradation activity of several dyes in the presence of H2O2 was tested, resulting in the detection of bromophenol blue-decolorizing activity. Based on these data, we suggest this enzyme is a novel enzyme with aflatoxin-degradation activity. Fluorescence measurements suggest that the enzyme cleaves the lactone ring of aflatoxin.     

Pluripotency of 17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus

Cochliobolus lunatus 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is pluripotent for several steroidal and nonsteroidal substrates. In the presence of NADPH the enzyme was found to reduce 3-keto groups of 4,5-dihydro steroids, 20-keto groups, and most efficiently, 17-keto groups of steroidal substrates. In addition, several quinones were accepted and found to be even better substrates as steroids due to their higher affinity for the enzyme-coenzyme complex and faster conversion of the enzyme-coenzyme-substrate complex into the corresponding products. As suggested by the competition studies quinones and 17-ketosteroids are converted by the same active center of the enzyme. For all tested substrates, the equilibrium ordered mechanism was established with NADPH binding first to the enzyme. According to our knowledge, the investigated 17beta-HSD is the first known fungal pluripotent enzyme of this type.     

Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.     

Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the nonpregrown cultures. In both culture conditions, P. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/l BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost the same as that in the suspended culture. The estrogenic activity of 100 mg/l DMP decreased during biodegradation by P. ostreatus.     

Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes

7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.     

The chitinolytic activity of Penicillium janthinellum P9: purification, partial characterization and potential applications

AIMS: To purify and characterize the chitinolytic activity of Penicillium janthinellum P9 and to evaluate possible uses of the purified enzymes in the control of fungal growth and spore germination. METHODS AND RESULTS: The chitinolytic activity of P. janthinellum P9 was associated to two beta-N-acetyl-hexosaminidases (CHI1 and CHI2) that were purified by preparative isoelectric focusing and preparative electrophoresis and partially characterized. Treatment of test fungi with purified enzyme solutions caused reduced spore germination, reduction of hyphal length and mycelial damage. The combined action of the two enzymes and a systemic fungicide completely inactivated pests and food-spoiling moulds such as Fusarium solanii, P. canescens and Cladosporium cladosporioides. Treatment with the two enzymes increased germination of freeze-dried fungal spores. CONCLUSION: The chitinolytic activity of P. janthinellum P9 is associated with two extracellular beta-N-acetyl-hexosaminidases that can cause damage to the cell walls of other fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This appears to be the first report on the characterization of extracellular chitinolytic enzymes produced by a Penicillium strain. The results of this study might have some impact in the applied research field.     

Novel inhibitors of 17beta-hydroxysteroid dehydrogenase type 1: templates for design

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone (E1) to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Syntheses and biological evaluation of novel non-steroidal inhibitors designed to mimic the E1 template are reported using information from potent steroidal inhibitors. Of the templates investigated biphenyl ethanone was promising and led to inhibitors with IC(50) values in the low micromolar range.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems.

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

Purification of 1,3-β-Glucan Synthase from Neurospora crassa by Product Entrapment

Awald, P., Zugel, M., Monks, C., Frost, D., and Selitrennikoff, C. P. 1993. Purification of 1,3-β-glucan synthase from Neurospora crassa by product entrapment. Experimental Mycology, 17, 130-141. 1,3-β-Glucan synthase activity of the ascomycete Neurospora crassa was purified ∼700-fold from hyphae. Hyphae were disrupted by bead-beating, and membrane-enriched fractions were obtained by high-speed centrifugation. Membranes were treated with (3-[(3-cholamidopropyl)dimethyl-ammoniol]I-propanesulfonate) and octyl-β-D-glucoside to solubilize enzyme activity. Soluble glucan synthase activity was incubated with substrate (UDP-glucose) and purified by centrifugation of enzyme associated with glucan (product entrapment). Purification was specific for UDP-glucose, the optimal concentration being 0.25 mM; no other nucleotide diphosphate sugar was able to significantly product-entrap enzyme activity. Partially purified enzyme activity formed β(1,3)-linked glucan, had a mean specific activity of 1900 nmol glucose incorporated/min/mg protein, a K_m,app of 0.7 mM, and a V_max of 0.5 nmol glucose incorporated/min. Separation of partially purified enzyme activity by SDS-PAGE showed a number of proteins copurifying with enzyme activity; computer analysis of digitized gel images revealed that proteins of 21, 25, 28, 45, 53, and 78 kDa were enriched. These results reinforce the view that 1,3-β-glucan synthase activity of fungi is a multimeric enzyme.