Functional and Structural Roles of the Dimer Interface in the Activity and Stability of Clostridium butyricum 1,3-Propanediol Oxidoreductase.

Abstract

Biosynthesis of 1,3-propanediol (1,3-PD) by 1,3-propanediol oxidoreductase (PDOR) is often limited by the stability issues. To address this issue, the goal of the present study was to engineer the Clostridium butyricum PDOR dimeric interface. The interface exists between the chains and plays a role in the synthesis of 1,3-PD, which is hindered by the increased temperature and pH. Herein, we engineered PDOR by HotSpot Wizard 3.0 and molecular dynamics simulations, improving its thermal stability, pH tolerance, and catalytic properties with respect to the wild-type PDOR activity at 37 °C. Compared to the activity of the wild-type PDOR, the N298C mutant showed 0.5-fold greater activity at pH 8.0, while the P299E mutant showed significantly increased activity of over five fold at pH 4.0. Further structural comparisons between the wild-type and P299E mutant revealed that the extraordinary stability of the P299E mutant could be due to the formation of additional hydrogen bonds and salt bridges. The N298C mutant also exhibits thermal stability at a broad range of temperature at pH 8 with respect to wild-type PDOR and other mutants. The molecular dynamics simulations revealed that stability profiles of P299E mutants at pH 4.0 are attributed to identical root mean square deviation values and stable conformations in the motif region present in the dimer interface of the enzyme. These findings suggest that the dimer interface motifs are essential for the compactness and stability of the PDOR enzyme; therefore, engineering the PDOR using a structure-guided approach could aid in improving its activity and stability under various physiological conditions (pH and temperature).