Litavadee ChuaboonThanyaporn WongnatePangrum PunthongCholpisit KiattiseweeNarin LawanChia Yi HsuChun Hung LinUwe T. BornscheuerPimchai ChaiyenAcademia Sinica, Institute of Biological ChemistryVidyasirimedhi Institute of Science and TechnologyErnst-Moritz-Arndt-Universität GreifswaldMahidol UniversityChiang Mai University2020-01-272020-01-272019-02-18Angewandte Chemie - International Edition. Vol.58, No.8 (2019), 2428-243215213773143378512-s2.0-85060784297https://repository.li.mahidol.ac.th/handle/20.500.14594/50521© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 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.Mahidol UniversityChemical EngineeringChemistryArticleSCOPUS10.1002/anie.201814219