Mutational and structural analysis of ribose 5-phosphate isomerase B from Acetivibrio thermocellus: relationship between transformation efficiency and substrate binding pocket conformation

D-allose is a rare hexose sugar with a variety of potential application in food, medicine and other fields. Ribose 5-phosphate isomerase（RPI） plays a pivotal role in the synthesis of D-allose. AtRpiB from Acetivibrio thermocellus can convert D-psicose to D-allose; however, improvements in its stability, optimal temperature, and conversion efficiency are necessary. This study aimed to investigate the catalytic properties, stability, and substrate-binding affinity of AtRpiB using D-psicose as the substrate. Using various strategies, 10 amino acid residues were selected to construct a library of 82 single mutants. Nine single mutants showed high conversion rates of D-allose. Furthermore, 27 double mutants were constructed by combining nine successful single mutation sites. Notably, the mutants R109W/R132Q, S39I/R109F, and S39V/R109F showed a 1.39-fold increase in enzyme activity at 40 ºC, 1.58-fold increase at 60 ºC, and 1.9-fold increase at 80 ºC, respectively. The S39V/R109F mutant converted 100 g/L D-psicose to 38.21 g/L D-allose after 18 hours at 40°C, the highest reported conversion rate for RpiB in D-allose production to date. Analysis of enzymatic characteristics, structure, and molecular dynamics simulations revealed that changes in the amino acid composition and conformational adjustments in loops 3, 9, and 10 of AtRpiB significantly affected the entry and exit of substrates and products into the active pocket, conversion efficiency, and enzyme stability.