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  • Title: The critical role of the N-terminus of 11beta-hydroxysteroid dehydrogenase type 1, as being encoded by exon 1, for enzyme stabilization and activity.
    Author: Blum A, Maser E.
    Journal: Chem Biol Interact; 2003 Feb 01; 143-144():469-80. PubMed ID: 12604233.
    Abstract:
    11beta-Hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders, including insulin resistance and obesity. Because 11beta-HSD 1 is a membrane protein with a very hydrophobic character, it is difficult to purify it in an active state. Not much is known about the topological and structural determinants of 11beta-HSD 1, although the elucidation of the structure of 11beta-HSD 1 would be a great advantage in identifying specific 11beta-HSD 1 inhibitors. Bacterial expression of full-length or truncated 11beta-HSD 1 forms only led to insoluble proteins or to low amounts of enzyme, not sufficient for crystallization. Recently, we reported that the solubility of 11beta-HSD 1 could be increased by substitution of hydrophobic amino acid residues with arginine without affecting activity. Unfortunately, these truncated and soluble forms of 11beta-HSD 1 exhibited an unstable activity that declined very rapidly. So far, the proteins obtained were not suitable for crystallization. To obtain 11beta-HSD 1 in an active and soluble state, in the present investigation we focused on the amino acid sequence encoded by the first exon. Using bacterial and yeast expression systems, we found that this N-terminal peptide could be divided into two parts that have functions other than to anchor 11beta-HSD 1 into the ER membrane. The first hydrophobic part, consisting of amino acid residues 1-15, represents the membrane spanning domain and anchors 11beta-HSD 1 in the ER membrane. The second hydrophilic part of the peptide, consisting of amino acid residues 16-30, plays a crucial role in stabilizing the catalytic domain of 11beta-HSD 1 and in addition, acts as a spacer to keep the catalytic domain of 11beta-HSD 1 into the lumen of the ER. Evidently, we found that the hydrophilic amino acids 24-30 determine 11beta-HSD 1 enzyme activity. Combined, all information obtained should help to design an optimal 11beta-HSD 1 enzyme in the near future with all desired attributes: soluble, active and easy to obtain and purify in sufficient amounts. This soluble and active 11beta-HSD 1 form should be the basis for our ongoing project, which is the determination of the three dimensional structure of 11beta-HSD 1.
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