title: The role of Ala231 and Trp227 in the substrate specificities of fungal 17β-hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase: Steroids versus smaller substrates.
abstract: 17β-Hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus; 17β-HSDcl and 3HNR, respectively) are two homologous short-chain dehydrogenase/reductase proteins that are 58% identical and have 86% similar amino acids. The minor differences in their substrate-binding regions are believed to be crucial for their substrate specificities. 3HNR shows high affinity for substrates with two rings, like trihydroxynaphthalene and 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), while 17β-HSDcl can accommodate ligands with four rings, like steroids. In the present study, we examined the role of Ala231 in 17β-HSDcl and Trp227 in 3HNR, as the potential key amino acids in the determination of substrate recognition based on size. We constructed Ala231Trp 17β-HSDcl and Trp227Ala 3HNR mutant proteins and used spectrophotometric analyses to compare their catalytic activities with those of the wild-type enzymes, for oxidation of 4-estrene-17β-ol-3-one and DDBO and for reduction of 4-estrene-3,17-dione and 9,10-phenanthrenequinone (PQ). The Ala231Trp side-chain substitution in 17β-HSDcl abolished and decreased (by 14.6-fold) the initial rates for steroid oxidation and reduction, respectively, while the initial rate for PQ reduction was increased 5.6-fold. The bulky Trp227Ala side-chain substitution in 3HNR enabled oxidation of 4-estrene-17β-ol-3-one, increased the initial rates for reduction of 4-estrene-3,17-dione and PQ by 4.5-fold and 1.5-fold, respectively, while the initial rate for DDBO oxidation was decreased 4.1-fold. Our TLC analysis and docking simulations also support these findings. Our study thus confirms the important roles of Ala231 in 17β-HSDcl and Trp227 in 3HNR, for the selection between larger and smaller substrates. Article from a special issue on steroids and microorganisms.
authors: Institute of Biochemistry, Ljubljana, Slovenia.
authors: Mojca Brunskole
authors: MB
authors: Svegelj
authors: Jure
authors: J
authors: Stojan
authors: Tea Lanišnik
authors: TL
authors: Rižner
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology; D001578:Benzopyrans / Q000378:metabolism; D020134:Catalytic Domain; D004963:Estrenes / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism; D013256:Steroids / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*
chemical_list: C111394:2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one; D001578:Benzopyrans; D004963:Estrenes; D005656:Fungal Proteins; D010616:Phenanthrenes; D013256:Steroids; C005399:9,10-phenanthrenequinone; D014364:Tryptophan; D015067:17-Hydroxysteroid Dehydrogenases; C086024:tetrahydroxynaphthalene reductase; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; D000409:Alanine