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  • Significance of individual amino acid residues for coenzyme and substrate specificity of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus
    Kristan, Katja, 1975- ...
    17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcI) is a NADPH dependent imember of the short-chain dehydrogenase reductase (SDR) superfamily. Recently, we ... prepared a homology-built structuralmodel of 17beta-HSDcI using the known three-dimensional structure ofhomologous 1,3,8-trihydroxynaphthalene reductase from the fungus Magnaporthegrisea. This model structure directed our studies of structure-function relationship of the fungal 17beta-HSD, as one of the model enzymes of the SDR superfamily. In this work, we investigated the significanceof individual amino acid residues for coenzyme and substrate specificity. We performed site directed mutagenesis of R28, a basic residue conserved in most NADPH dependent SDR structures; T200, found only in Streptomyces hydrogenans 3alpha, 20beta-HSD and Drosophila alcohol dehydrogenases; and H230, a residue corresponding to the substrate specificityimportant H221 in human 17beta-HSD type 1. All recombinant proteinswere expressed in Escherichia coli and purified to homogeneity. Kinetic evaluation of individual mutations was performed by analysis of progress curves of interconversions between 4-estrene-3,17-dione and 4-estrene-17beta-ol-3-one, in the presence of NADPH and NADP+; according to the Theorell-Chance reaction mechanism. The results demonstrate the role of the selected amino acid residues; R28 seems to interact with the NADPH 2'-phosphate group; T200 may be involved in binding and dissociation of NADPH/NADP+; while H230 and the neighboring A231 appears not to be responsiblefor substrate specificity of 17beta-HSDcI.
    Vir: Chemico-biological interactions. - ISSN 0009-2797 (št. 143/144, 2003, str. 493-501)
    Vrsta gradiva - članek, sestavni del
    Leto - 2003
    Jezik - angleški
    COBISS.SI-ID - 15924441