Expression of human kidney 11beta-hydroxysteroid dehydrogenase (11-HSD2) in bacteria.
The kidney isozyme of 11beta-hydroxysteroid dehydrogenase (11-HSD2) protects the mineralocorticoid receptor from spurious activation by glucocorticoids. To explore structure-function relationships, human 11-HSD2 cDNA was subcloned into the bacterial expression vector, pET25b. E. coli transformed with wild-type cDNA produced active enzyme that retained biochemical characteristics of the native protein. The addition of 6 histidine residues to the C-terminus of the wild-type enzyme (11-HSD2/His) increased activity 2-fold. Whereas wild-type activity was almost completely sedimented following 100,000g centrifugation, 10-30% of total activity of 11-HSD2/His remained in the supernatant. The 11-HSD2 isozyme normally contains three N-terminal hydrophobic domains. Mutant 11-HSD2/His possessing a single hydrophobic domain retained partial activity, but elimination of all domains inactivated the enzyme. Thus, the N-terminal hydrophobic domains are essential for complete activity of 11-HSD2 but association with an intact cell membrane is not.
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, 75235-9063, USA.
B S
BS
Nunez
T
T
Mune
P C
PC
White
D042842:11-beta-Hydroxysteroid Dehydrogenases; D004790:Enzyme Induction / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014461:Ultracentrifugation
D007527:Isoenzymes; D011994:Recombinant Proteins; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases