One-Pot Bioconversion of l-Arabinose to l-Ribulose in an Enzymatic Cascade
Graphical Abstract
A spoonful of sugar: A new concept for the synthesis of l-ribulose from l-arabinose was successfully established using a one-pot bioconversion consisting of engineered pyranose 2-oxidase, xylose reductase, formate dehydrogenase, and catalase. Transient kinetics in combination with molecular dynamics simulations indicate that the sugar substrate is positioned closer to the FAD N5-position in the engineered pyranose 2-oxidase than in the wild-type.
Abstract
This work reports the one-pot enzymatic cascade that completely converts l-arabinose to l-ribulose using four reactions catalyzed by pyranose 2-oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild-type P2O is specific for the oxidation of six-carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l-arabinose at the C2-position. The variant T169G was identified as the best candidate, and this had an approximately 40-fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild-type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4-OH of l-arabinose, which allows a reduction in the distance between the C2-H and flavin N5, facilitating hydride transfer and enabling flavin reduction.
