RESUMO
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 α-, ß-, γ- and δ-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 ß-subunit C-terminal residues. Reduction of flavin cofactors shifts the ß-subunit Ala1 towards the α-subunit Met55, forming a surface cavity at the oxygen-channel vestibule and rendering the ß-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 α and ß-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 ß-subunit C-terminal residues. The third channel goes through the α-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 H(2)O(2) formed exits via the oxygen channel.
Assuntos
Corynebacterium/enzimologia , Conformação Proteica , Domínio Catalítico , Cristalografia por Raios X , Leucovorina/química , Leucovorina/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sarcosina Oxidase/química , Sarcosina Oxidase/genética , Sarcosina Oxidase/metabolismo , Tetra-Hidrofolatos/química , Tetra-Hidrofolatos/metabolismoRESUMO
Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The alpha subunit is composed of two domains, contains NAD(+), and binds folinic acid. The beta subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10A apart. The gamma subunit is in contact with two domains of alpha subunit and has possibly a folate-binding structure. The delta subunit contains a single atom of zinc and has a Cys(3)His zinc finger structure. Based on the structures determined and on the previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.
