Channeling and conformational changes in the heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

We characterized the crystal structures of heterotetrameric sarcosine oxidase (SO) from Corynebacterium sp. U-96 complexed with methylthioacetate (MTA), pyrrole 2-carboxylate (PCA) and sulphite, and of sarcosine-reduced SO. SO comprises alpha-, beta-, gamma- and delta-subunits; FAD and FMN cofactors; and a large internal cavity. MTA and PCA are sandwiched between the re-face of the FAD isoalloxazine ring and the beta-subunit C-terminal residues. Reduction of flavin cofactors shifts the beta-subunit Ala1 towards the alpha-subunit Met55, forming a surface cavity at the oxygen-channel vestibule and rendering the beta-subunit C-terminal residues mobile. We identified three channels connecting the cavity and the enzyme surface. Two of them exist in the inter-subunit space between alpha and beta-subunits, and the substrate sarcosine seems to enter the active site through either of these channels and reaches the re-side of the FAD isoalloxazine ring by traversing the mobile beta-subunit C-terminal residues. The third channel goes through the alpha-subunit and has a folinic acid-binding site, where the iminium intermediate is converted to Gly and either formaldehyde or, 5,10-methylenetetrahydrofolate. Oxygen molecules are probably located on the surface cavity and diffuse to the FMN isoalloxazine ring; the H2O2 formed exits via the oxygen channel.