A novel 17β-hydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the evolution of steroid-hormone signalling

Abstract

17β-Hydroxysteroid dehydrogenase (17β-HSD) from the filamentous fungus Cochliobolus lunatus (17β-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 17β-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. 17β-HSDcl is the first discovered fungal 17β-hydroxysteroid dehydrogenase belonging to this family. The primary structure of this enzyme may therefore help to elucidate the evolutionary history of steroid dehydrogenases.

Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

“…Among the tens of SDR enzymes for which the three-dimensional structures have been resolved, only two are known to be active in solution as monomers (pdb code 1N5D and 1WMA) [ 6 , 7 ]. We have shown previously, that the wild-type 17β-HSDcl acts as dimer [ 10 ]. In order to influence the formation of this dimer, we substituted two surface amino acids which should not, per se , prevent either the folding or the activity.…”

“…The mutant proteins were prepared using the Quick-Change Site-Directed Mutagenesis Kit (Stratagene) and the pGex-17β-HSDcl expression vector [ 10 ]. The following primers were used (only mutagenic forward primers are shown; the mutations introduced are underlined):…”

“…Although there are about three thousand members of the SDR superfamily in the species studied [ 8 ], 17β-HSD from the fungus Cochliobolus lunatus (17β-HSDcl) [ 9 ] is currently the only fungal HSD member that has been characterized. The enzyme has been purified, and also cloned and expressed in E. coli [ 9 , 10 ]. Under native conditions, both recombinant [ 10 ] and natural [ 9 ] 17β-HSDcl form dimers.…”

“…The enzyme has been purified, and also cloned and expressed in E. coli [ 9 , 10 ]. Under native conditions, both recombinant [ 10 ] and natural [ 9 ] 17β-HSDcl form dimers. 17β-HSDcl is homologous to fungal reductases: to versicolorin reductases from Aspergillus parasiticus and Emericella nidulans , which are involved in aflatoxin biosynthesis; and to the 1,3,8-trihydroxynaphthalene reductases and 1,3,6,8-tetrahydroxynaphthalene reductases (3HNR, 4HNR) from Magnaporthe grisea , Ophiostoma floccosum and other fungi, which are involved in melanin biosynthesis [ 11 ].…”

The HOG pathway in the halophilic black yeast Hortaea werneckii: isolation of the HOG1 homolog gene and activation of HwHog1p

“…A HOG1-harboring DNA fragment (approximately 580 bp) was obtained by polymerase chain reaction (PCR) ampli¢cation with oligonucleotides 5P-ACGGAATTA-CAAGGAACAGATTTAC-3P and 5P-GCAGTGATTCT-CTTCTTAGGATC-3P as primers and S. cerevisiae genomic DNA as the template DNA. The PCR was performed as described elsewhere [29], with an annealing temperature of 56 ‡C. After puri¢cation by agarose electrophoresis and Qiaquick PCR Puri¢cation Kits (Qiagen), the DNA fragment was labeled with [ 32 P]dCTP using the Prime-It 0 RmT Random Primer Labeling Kit (Stratagene) according to the manual.…”