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  • Title: Characterization of truncated mutants of human microsomal short-chain dehydrogenase/reductase RoDH-4.
    Author: Belyaeva OV, Chetyrkin SV, Kedishvili NY.
    Journal: Chem Biol Interact; 2003 Feb 01; 143-144():279-87. PubMed ID: 12604214.
    Abstract:
    Human NAD(+)-dependent microsomal short-chain dehydrogenase/reductase RoDH-4 oxidizes all-trans-retinol, 13-cis-retinol and 3alpha-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 3alpha-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 approximately 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.
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