Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric
flavin-dependent oxidase that performs selective oxidation of the terminal
primary hydroxyl group of several alditols. Here, we report the crystal
structure of the recombinant enzyme in its native state and in complex with both
six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO
shares the same folding topology of the members of the vanillyl-alcohol oxidase
family of flavoenzymes and exhibits a covalently linked FAD which is located at
the bottom of a funnel-shaped pocket that forms the active site. The high
resolution of the three-dimensional structures highlights a well-defined
hydrogen-bonding network that tightly constrains the substrate in the productive
conformation for catalysis. Substrate binding occurs through a lock-and-key
mechanism and does not induce conformational changes with respect to the
ligand-free protein. A network of charged residues is proposed to favor
catalysis through stabilization of the deprotonated form of the substrate. A His
side chain acts as back door that "pushes" the substrate-reactive carbon atom
toward the N5-C4a locus of the flavin. Analysis of the three-dimensional
structure reveals possible pathways for diffusion of molecular oxygen and a
small cavity on the re side of the flavin that may host oxygen during FAD
reoxidation. These features combined with the tight shape of the catalytic site
provide insights into the mechanism of AldO-mediated regioselective oxidation
reactions and its substrate specificity.
