One‐Pot Bioconversion of L‐Arabinose to L‐Ribulose in an Enzymatic Cascade

This work reports the first one‐pot enzymatic cascade which completely converts l‐arabinose to l‐ribulose using four reactions catalyzed by pyranose 2‐oxidase (P2O), xylose reductase, formate dehydrogenase and catalase. As the wild‐type P2O is specific for oxidation of six‐carbon sugars such as d‐glucose, a pool of P2O variants were generated based on rational design in order to change the specificity of the enzyme towards oxidation of l‐arabinose at the C2‐position. The variant T169G was identified as the best candidate, and investigations using transient kinetics indicated that this variant had an approximately 40‐fold higher rate constant for the flavin reduction (sugar oxidation) step and about 13‐fold increase in kcat/Km, as compared to the wild‐type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) indicated 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 efficient flavin reduction.

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