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Roles of interfacial interactions of D249 and R167 in the oligomerization of 3α‐hydroxysteroid dehydrogenase/carbonyl reductase
Author(s) -
Hwang ChiChing,
Huang TzuJung,
Hsu ChaoNan,
Wang YuanLiang
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.466.2
Subject(s) - chemistry , salt bridge , enzyme , dehydrogenase , urea , mutant , protein subunit , size exclusion chromatography , equilibrium unfolding , thermal stability , crystallography , biochemistry , stereochemistry , protein folding , organic chemistry , gene
The dimerization of 3α‐hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. In this study, we explored the mechanisms of dimerization and stabilization in the 3α‐HSD/CR. Mutants of D249A, D249K, D249S, R167A, R167D, and R167Q 3α‐HSD/CRs were constructed, and their oligomeric forms were characterized through gel filtration chromatography. Enzyme catalysis was studied by kinetic analysis. Protein stability was studied through thermal and urea unfolding by CD spectroscopy. Mutation on D249 and R167 causes different effects on the loss of enzymatic activity and shows a concentration‐dependent dimerization of wide‐type and mutant enzymes. Thermal and urea‐induced unfolding profile for wild‐type and mutant enzymes appeared as a two‐state transition and three‐state transition, respectively. The study from the thermal and urea unfolding indicates the mutation of the residue D249 and R167 destabilize the protein structure. Therefore, the salt‐bridge interaction between D249 and R167 of each subunit is important in stabilizing dimeric formation of 3α‐HSD/CR.