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

Abstract

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.

Mutations that affect coenzyme binding and dimer formation of fungal 17β-hydroxysteroid dehydrogenase

“…2). 17β-HSDcl is dimer under native conditions (Lanišnik Rižner et al, 1999). Structural model (Lanišnik Rižner et al, 2000) and a recently resolved crystal structure (our unpublished data) identified salt bridges/H-bonds (between R129 and E117/D121, and between H111 and D187) and strong hydrophobic and aromatic interactions (for F120, F124, F132, F133, F177) among the αE and αF helices of both of the subunits that can stabilize dimerization (Kristan et al, 2005b).…”

“…We have shown previously that wild-type 17β-HSDcl is active as a dimer (Lanišnik Rižner et al, 1999). Examination of the three-dimensional model and crystal structure of fungal 17β-HSD (Lanišnik Rižner et al, 2000; our unpublished data) has allowed the identification of salt bridges/ H-bonds (H111, E117, D121, R129, D187) and strong hydrophobic and aromatic interactions (F120, F124, F132, F133, F177) across the Q-axis.…”

“…A c c e p t e d M a n u s c r i p t 3 The 17β-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) was the first fungal SDR to be purified and cloned (Lanišnik Rižner et al, 1999). It is an NADPH-preferring enzyme and it preferentially catalyses reversible oxidoreductions of androgens and estrogens at the C17 position (Lanišnik Rižner et al, 2000, Kristan et al, 2003.…”

“…It is an NADPH-preferring enzyme and it preferentially catalyses reversible oxidoreductions of androgens and estrogens at the C17 position (Lanišnik Rižner et al, 2000, Kristan et al, 2003. 17β-HSDcl contains all of the typical motifs of the classical SDR family (Lanišnik Rižner et al, 1999). Two threonines (T202 and T200) from the [KR]x[NS]xxxPGxT (200) xT (202) motif are in close contact with the nicotinamide moiety, and we have already studied their role for interactions with NADP(H) (Kristan et al, 2003, Kristan et al, 2005a).…”

“…The following primers were used (only forward primers are shown, with the mutations introduced underlined): M204G: 5'-CGGTACCGTGACAGATGGCTTCCACGAGGTCTCG-3' F124A: 5'-GAGTTTGACCGCGTCGCGAGCCTCAACACCCG-3' F132A: 5'-CCCGAGGCCAGGCGTTCGTCGCTCGC-3' F132/133A: 5'-CCCGAGGCCAGGCGGCGGTCGCTCGCGAGGC -3' F177A: 5'-GCCGTCGACTCCGCGGTTCGCATTTTCTCAAAGG -3' All of the proteins were prepared as a glutathione S-transferase (GST)-fusion proteins in BL21-AI Escherichia coli cells (Invitrogen) and purified by affinity binding to glutathione-Sepharose, followed by cleavage with thrombin, as described previously (Lanišnik Rižner et al, 1999). The purities of the proteins were checked by SDS-PAGE.…”