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Litavadee Chuaboon Thanyaporn Wongnate Pangrum Punthong Cholpisit Kiattisewee Narin Lawan Chia‐Yi Hsu Chun‐Hung Lin Uwe T. Bornscheuer Pimchai Chaiyen 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2019,131(8):2450-2454
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. 相似文献
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Thanyaporn Wongnate Panida Surawatanawong Litavadee Chuaboon Narin Lawan Pimchai Chaiyen 《Chemistry (Weinheim an der Bergstrasse, Germany)》2019,25(17):4460-4471
Understanding the reaction mechanism underlying the functionalization of C−H bonds by an enzymatic process is one of the most challenging issues in catalysis. Here, combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C−H bond oxidation in d -glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O). Unlike the mechanisms that have been conventionally proposed, our findings show that the first step of the C−H bond oxidation reaction is a hydride transfer from the C2 position of d -glucose to N5 of the flavin to generate a protonated ketone sugar intermediate. The proton is then transferred from the protonated ketone intermediate to a conserved residue, His548. The results show for the first time how specific interactions around the sugar binding site promote the hydride transfer and formation of the protonated ketone intermediate. The DFT results are also consistent with experimental results including the enthalpy of activation obtained from Eyring plots, as well as the results of kinetic isotope effect and site-directed mutagenesis studies. The mechanistic model obtained from this work may also be relevant to other reactions of various flavoenzyme oxidases that are generally used as biocatalysts in biotechnology applications. 相似文献
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