Enhanced isomerization of rare sugars by ribose-5-phosphate isomerase A from Ochrobactrum sp. CSL1

Ribose-5-phosphate isomerase A (RpiA) is of great importance in biochemistry research, however its application in biotechnology has not been fully explored. In this study the activity of RpiA from Ochrobactrum sp. CSL1 (OsRpiA) towards D-allose was engineered based on sequential and structural analyses. Strategies of alanine scanning, rational design and saturated mutagenesis were employed to create three mutant libraries. A single mutant of K124A showed a 45 % activity improvement towards D-allose. The reaction properties of the mutant were analyzed, and a shift of optimal pH and higher thermal stability at low reaction temperatures were identified. The conversion of D-allose was also improved by 40 % using K124A, and higher activities on major substrates were found in the mutant's substrate scope, implying its application potential in rare sugar preparation. Kinetics analysis revealed that K_m of K124A mutant decreased by 12 % and the catalytic efficiency increased by 65 % towards D-allose. Moreover, molecular dynamics simulation illustrated the binding of substrate and K124A was more stable than that of the wild-type. The shorter distance and more relax bond angle between the catalytic residue of K124A and D-allose explained the activity improvement in detail. This study highlights the potential of OsRpiA as a biocatalyst for rare sugar preparation, and provides distinct evidences for its catalytic mechanism.