Interactions across the interface contribute the stability of homodimeric 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase.
The dimerization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 degrees C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3alpha-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.
Hwang
Chi-Ching
CC
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80731, Taiwan. cchwang@kmu.edu.tw
Hsu
Chao-Nan
CN
Huang
Tzu-Jung
TJ
Chiou
Shean-Jaw
SJ
Hong
Yi-Ren
YR
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000235:genetics* / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D002384:Catalysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D012694:Serine / Q000235:genetics; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic; D014508:Urea / Q000494:pharmacology
D012694:Serine; D014508:Urea; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D008239:Lysine; D000409:Alanine