Exploring Multifunctional Residues of Ribose-5-phosphate Isomerase B from Ochrobactrum sp. CSL1 Enhancing Isomerization of d-Allose.

Ribose-5-phosphate isomerase B is of great importance for biocatalysis and biosynthesis, but the multifunctional residues in active sites hinder the research efforts. This study employed rational design strategies to locate the key residues of RpiB from Ochrobactrum sp. CSL1 (OsRpiB). A single-mutant S9T of a noncontact residue showed 80% activity improvement toward d-allose. A double-mutant S98H/S134H further increased the activity to 3.6-fold. The mutations were analyzed by kinetics and molecular dynamics analyses, indicating that S9T might enhance the substrate binding and catalysis by inducing a steric effect, and S98H/S134H could strengthen both ring opening and binding of d-allose. Though S98H/S134H showed low temperature stability, its potential was explored by isomerizing d-allose to d-psicose with higher conversion and in less reaction time. The findings of this study were beneficial for illustrating the complex functions of key residues in RpiBs and applying OsRpiB in preparing rare sugars.