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  • The role of Ala231 and Trp227 in the substrate specificities of fungal 17ß-hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase : steroids versus smaller substrates
    Brunskole Švegelj, Mojca ; Stojan, Jure, 1956- ; Lanišnik-Rižner, Tea
    17beta-Hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus; 17beta-HSDcl and 3HNR, respectively) are two homologous ... short-chain dehydrogenase/reductaseproteins 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 17beta-HSDcl can accommodate ligands with four rings, like steroids. In the present study, we examined the role of Ala231 in 17beta-HSDcl and Trp227 in 3HNR, as the potential key amino acids in the determination of substrate recognition basedon size. We constructed Ala231Trp 17beta-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-17beta-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 17beta-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-17beta-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 17beta-HSDcl and Trp227 in 3HNR, for the selection between larger and smaller substrates.
    Vrsta gradiva - članek, sestavni del
    Leto - 2012
    Jezik - angleški
    COBISS.SI-ID - 28211673