Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase

• Published in: Molecules and cells 2013, 36(1), , pp.39-46
• Main Authors: Cha, H.J., Pohang University of Science and Technology, Pohang, Republic of Korea, Jang, D.S., Research Institute, Genexine Co., Seongnam, Republic of Korea, Kim, Y.G., Pohang University of Science and Technology, Pohang, Republic of Korea, Hong, B.H., Research Institute, Genexine Co., Seongnam, Republic of Korea, Woo, J.S., Pohang University of Science and Technology, Pohang, Republic of Korea, Kim, K.T., Pohang University of Science and Technology, Pohang, Republic of Korea, Choi, K.Y., Pohang University of Science and Technology, Pohang, Republic of Korea
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Biocatalysis - drug effects; Biochemistry; Biomedical and Life Sciences; Biomedicine; Biotechnology; Catalytic Domain; Cell Biology; Crystallography, X-Ray; Enzyme Stability - drug effects; Hydrogen Bonding - drug effects; ISOMERASAS; ISOMERASE; ISOMERASES; Isomerism; ketosteroid isomerase,more hydrophobic interactions,rescue mechanism,second-site suppressor; Kinetics; Life Sciences; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation - genetics; Protein Folding - drug effects; Pseudomonas putida - enzymology; Steroid Isomerases - genetics; Urea - pharmacology; 생물학
• ISSN: 1016-8478; 0219-1032
• DOI: 10.1007/s10059-013-0013-1

Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a secondsite suppressor.