Modification of galactose oxidase to introduce glucose 6-oxidase activity.

The selective oxidation of the 6-hydroxy group of D-glucose to introduce an aldehyde functionality is not catalyzed by known oxidase enzymes. Selective functionalization at the glucose C-6 position in oligoand polysaccharides is a synthetically useful reaction that would greatly facilitate further chemical modifications for food, pharmaceutical, and materials applications. We chose the fungal enzyme galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) as a starting point to try to generate a glucose 6-oxidase. A well-characterized copper-containing radical enzyme, galactose oxidase (GOase) oxidizes various primary alcohols to their corresponding aldehydes, with the reduction of oxygen to hydrogen peroxide. Native GOase is highly active towards the 6-OH group of D-galactose but essentially inactive towards D-glucose. Glucose apparently cannot bind at the active site since concentrations as high as 1M have no effect on the activity of the enzyme towards Dgalactose. We first attempted to generate activity towards D-glucose by random point mutagenesis. After screening more than 30000 clones, however, we observed no improvement in D-glucose activity, although both the expression level and thermostability were enhanced. Careful examination of GOase activity shows that it is at least one million times less active towards D-glucose than towards D-galactose. We therefore concluded that the desired novel activity would require significant remodeling of the active site and is not accessible by point mutagenesis. The crystal structure of GOase has been solved and a substrate binding model has been proposed based on a molecular docking experiment. 5] According to the model, Arg330 forms hydrogen bonds with the hydroxy groups of substrate C-4 and C-3 atoms, while Gln406 forms an additional hydrogen bond with the C-2 hydroxy group of the substrate. In addition, a hydrophobic wall in the pocket that contains Phe194 and Phe464 interacts with the D-galactose backbone atoms C-6, C-5, and C-4. Trp290, which has been proposed to stabilize the radical form of GOase, is believed to play a key role in restricting entry to the active center (Figure 1).