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

Abstract

This work reports the one-pot enzymatic cascade that completely converts l-arabinose to l-ribulose using four reactions catalyzedb yp yranose 2-oxidase( P2O), xylose reductase,f ormate dehydrogenase,a nd catalase.A sw ild-type P2O is specific for the oxidation of six-carbon sugars,apool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of larabinose at the C2-position. The variant T169G was identified as the best candidate,a nd this had an approximately 40-fold higher rate constant for the flavin reduction (sugar oxidation) step,a sc ompared to the wild-type enzyme.C omputational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to ad ecrease in the steric effects at the axial C4-OH of l-arabinose,w hicha llows ar eduction in the distance between the C2-H and flavin N5, facilitating hydride transfer and enabling flavin reduction.