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

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 var...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2019-02, Vol.58 (8), p.2428-2432
Main Authors: Chuaboon, Litavadee, Wongnate, Thanyaporn, Punthong, Pangrum, Kiattisewee, Cholpisit, Lawan, Narin, Hsu, Chia‐Yi, Lin, Chun‐Hung, Bornscheuer, Uwe T., Chaiyen, Pimchai
Format: Article
Language:English
Subjects:
Arabinose
Bioconversion
Cascade chemical reactions
Catalase
Chemical reactions
Computer applications
enzymatic cascades
Enzymes
Flavin
flavoenzyme
Formate dehydrogenase
Molecular dynamics
one-pot reaction
oxidase
Oxidation
Quantum mechanics
Reduction
ribulose
Steric effects
Sugar
Xylose
Xylose reductase
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Summary: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. 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.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201814219