Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain.

1999 
Human 3‚-hydroxysteroid dehydrogenase/steroid ˜ 5 -˜ 4 -isomerase (3‚-HSD/isomerase) is a bifunctional, single enzyme protein that is membranebound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72‐89 and 283‐310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. DiVerential centrifugation of the Sf9 cell homogenate containing the 283‐310 deletion mutant revealed that 94% of the 3‚-HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72‐89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/ mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283‐310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383‐310 deletion mutant exhibited 3‚-HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283‐311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72‐89 deletion mutant had low 3‚-HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283‐310 region as the critical membrane domain of 3‚-HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72‐89 region is also a membrane domain, but deletion of this NH2-terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283‐310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/ quaternary structure of this key steroidogenic
    • Correction
    • Source
    • Cite
    • Save
    • Machine Reading By IdeaReader
    17
    References
    21
    Citations
    NaN
    KQI
    []