Truncation of the N- and C-terminal regions of the human 11beta-hydroxysteroid dehydrogenase type 2 enzyme and effects on solubility and bidirectional enzyme activity.
The 11beta-hydroxysteroid dehydrogenase type II enzyme (11betaHSD2) endows specificity on the mineralocorticoid receptor by metabolising glucocorticoids. Sequence comparisons with other microsomal proteins showed the strongly preferred topology of a lumenal pentapeptide followed by three transmembrane helices with residues beyond Ala73 on the cytoplasmic side of the membrane, suggesting that 11betaHSD2 is anchored to the endoplasmic reticulum by the N-terminal region. However, deletion of the N-terminus (11betaHSD2 deltaN) and expression of the construct in mammalian cells showed that the enzyme remained bound to the microsomal fraction, indicating that other regions are also involved in membrane anchoring. Crosslinking studies and nonreducing SDS-PAGE demonstrated that 11betaHSD2 is a non-covalently linked dimer. Deletion of the non-conserved C-terminal region (11betaHSD2 deltaC) resulted in an enzyme with a Km of 215 nM for cortisol in whole cell assays, while 11betaHSD2 and 11betaHSD2 deltaN displayed a Km of 62 and 74 nM, respectively. In homogenates 11betaHSD2 and 11betaHSD2 deltaC displayed maximal activity at 140 mM NaCl or KCl, but showed a marked decrease in enzyme activity with increasing salt. 11BetaHSD2 was more stable than 11betaHSD2 deltaC in the presence of NaSCN, suggesting that the C-terminal region plays a role in enzyme stability. There was no detectable activity in homogenates containing 11betaHSD2 deltaN, while 11betaHSD2 deltaC and 11betaHSD2 displayed a Km of 135 and 46 nM, respectively. Although 11betaHSD2 is conventionally considered a unidirectional dehydrogenase all constructs converted 11-dehydrodexamethasone to dexamethasone in whole cell assays, providing an explanation for the potency of the synthetic glucocorticoid in the face of a powerful inactivator of natural glucocorticoids.
Obeyesekere
V R
VR
Laboratory of Molecular Hypertension, Baker Medical Research Institute, Prahran, Australia.
Li
K X
KX
Ferrari
P
P
Krozowski
Z
Z
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012995:Solubility; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D010446:Peptide Fragments; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D006854:Hydrocortisone