Identification of a gatekeeper residue that prevents dehydrogenases to act as oxidases

The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in L-galactono-1,4-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala113Gly) resulted in a striking 400 fold increase in oxygen reactivity, while the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (kox = 3.5 x 10e5 M-1 s-1), similar to most other flavoprotein oxidases. The Ala113Gly replacement does not change the reduction potential of the flavin, but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme Ala113 acts as a gatekeeper, preventing oxygen to access the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family, and is absent in members that act as oxidases.