4K1U

Crystal structure of delta5-3-ketosteroid isomerase containing Y16F and Y32F mutations

Summary for 4K1U

• Entry DOI: 10.2210/pdb4k1u/pdb
• Related: 1OPY 4K1V
• Descriptor: Steroid Delta-isomerase (2 entities in total)
• Functional Keywords: cystatin-like fold, allylic isomerization, delta5-3ketosteroids, cytosol, isomerase
• Biological source: Pseudomonas putida
• Total number of polymer chains: 2
• Total formula weight: 29033.00

Authors

Cha, H.J.,Jang, D.S.,Kim, Y.G.,Hong, B.H.,Woo, J.S.,Choi, K.Y. (deposition date: 2013-04-05, release date: 2013-07-31, Last modification date: 2024-03-20)

Primary citation

Cha, H.J.,Jang, D.S.,Kim, Y.G.,Hong, B.H.,Woo, J.S.,Kim, K.T.,Choi, K.Y.
Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase
Mol.Cells, 36:39-46, 2013

PubMed Abstract: 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.

PubMed: 23740430
DOI: 10.1007/s10059-013-0013-1

Experimental method

X-RAY DIFFRACTION (2 Å)