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

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

Angew Chem Int Ed Engl. 2019 Feb 18;58(8):2428-2432
doi: 10.1002/anie.201814219

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摘要

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.