Three-Dimensional Structures of a Wild-Type Ketosteroid Isomerase and Its Single Mutant in Solution

Abstract

The three-dimensional structures of the wild-type Pseudomonas putida ketosteroid isomerase (KSI-WT) and its single mutant KSI-Y57S were studied in solution for the first time using synchrotron X-ray scattering methods. The protein solutions were further analyzed using pulse field gradient nuclear magnetic resonance spectroscopy and ultracentrifugation analysis. The X-ray scattering profiles were extracted from the atomic crystal structures and analyzed. The KSI-WT solution structure differed in size and shape from the structure observed in the crystal form. In solution, the KSI-WT structure included an open cavity around the active site. This feature might be essential for the catalytic performance. The structure of KSI-WT changed upon introduction of a single mutation (the Tyr57 of KSI-WT was replaced with a serine residue). This single mutation was found to reduce the size and shape of the KSI-WT structure. However, the open cavity around the active site was abnormally enlarged in the presence of the single mutation. The changes in the size and shape might directly correlate with the large decrease in the catalytic performance of KSI-Y57S.