Characterization of truncated mutants of human microsomal short-chain dehydrogenase/reductase RoDH-4

• Published in: Chemico-biological interactions 2003-02, Vol.143, p.279-287
• Main Authors: Belyaeva, Olga V., Chetyrkin, Sergei V., Kedishvili, Natalia Y.
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Animals; Base Sequence; Catalysis; Dehydrogenase; DNA Primers; Human; Humans; Hydroxysteroid; Microsomes - enzymology; Mutation; Plasmids; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Retinol; Spodoptera; Subcellular Fractions - metabolism; Topology
• ISSN: 0009-2797; 1872-7786
• DOI: 10.1016/S0009-2797(02)00181-3

Human NAD +-dependent microsomal short-chain dehydrogenase/reductase RoDH-4 oxidizes all- trans-retinol, 13- cis-retinol and 3α-hydroxysteroids to corresponding retinaldehydes and 3-ketones. RoDH-4 behaves as an integral membrane protein, but its topology in the membrane is not known. Analysis of RoDH-4 polypeptide using algorithms for secondary structure predictions suggests that RoDH-4 contains four potential membrane-spanning domains: the N-terminal, the C-terminal, and the two central hydrophobic segments. To determine the role of each segment in association of RoDH-4 with the membrane, we prepared several expression constructs coding for truncated RoDH-4 polypeptides that lacked the putative membrane-spanning domains and expressed them in insect Sf9 cells using the Baculovirus system. Association of truncated RoDH-4 constructs with the microsomal membranes was analyzed by alkaline extraction and floatation in sucrose gradient. Catalytic activity of truncated RoDH-4 constructs was assayed using the 3α-hydroxysteroid androsterone as substrate. Truncated RoDH-4 that lacked the first thirteen amino acids of the N-terminal segment was partially active and exhibited the apparent K m value for androsterone similar to that of the wild-type enzyme. Removal of 23 N-terminal hydrophobic amino acids resulted in significant loss of activity and a 14-fold increase in the apparent K m value. Removal of the C-terminal 27 amino acid segment resulted in a ∼600-fold increase in the apparent K m value. Each truncated mutant behaved as an integral membrane protein. Furthermore, protein that lacked all four hydrophobic segments remained associated with the membrane. Thus, the N-terminal and the C-terminal ends are both important for RoDH-4 activity and the removal of the putative transmembrane segments does not convert RoDH-4 into a soluble protein, suggesting additional sites of membrane interaction.