On the role of double covalent flavin binding in chitooligosaccharide oxidase from Fusarium graminearum
Résumé
Chitooligosaccharide oxidase (ChitO) from Fusarium graminearum catalyzes the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site directed mutagenesis. The double mutant (H94A/C154A) was not expressed which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants (H94A and C154A) were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to wild-type enzyme. Both mutants were poorly active as the kcat decreased (8.3 and 3 fold, respectively) and the KM increased drastically (34 and 75 fold, respectively) when using N-acetyl-D-glucosamine as substrate. Pre-steady state analysis revealed that the rate of reduction in the mutant enzymes is reduced by 3 orders of magnitude when compared with wild-type ChitO (kred = 750 s-1) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV), the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.
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