Fig. 3: Overall structures of Orf1.

a Orf1 is a tetrameric complex of a dimer of dimers in solution, of which each protomer contains a FAD prosthetic group. b Each protomer is comprised of three domains, a FAD binding ___domain (red), a substrate binding ___domain (green) and a C-terminal ___domain (blue). The tetrameric complex is interfaced by the substrate binding ___domain and the C-terminal ___domain. c The glycine binding site is located on the top of the re face of the isoalloxazine ring of FAD and shaped by residues A54, G55, A56, M57, Y294, H284, R342, and R368 revealed from the Orf1-glycine complex. d Sarcosine also binds to the glycine-binding site as revealed from the Orf1-sacrosine complex. e R368 is misplaced when R342 was mutated to Ala resulting in the loss of enzyme activity as revealed from the Orf1-R342A structure. f The surface presentation of the glycine binding pocket, where a cavity nearby the active site was speculated the glycylthricin binding site. g The complex structure of Orf1-glycine-glycylthricin. Each Orf1 protomer in the tetrameric complex contains one molecule of FAD, glycine and glycylthricin. h The glycylthricin binding site is located at the inter-subunit junctions, where binding pockets are formed. i The 2F_o-F_c density map of glycylthricin is countered at 1 σ shown in a gray mesh. j The distance between the amine nitrogen atom of glycylthricin and the α-carbon of glycine is 13.5 Å away from each other. The antiparallel-β-sheet (β11- β15) acts as a physical barrier that delimits a boundary for both the glycylthricin and glycine binding pockets. k Superposition of crystal structures of wildtype and mutants in complex with glycylthricin. The Orf1-glycylthricin (B chain), C281S-glycylthricin (B chain), F316A-glycylthricin (B chain), R342A-glycylthricin (A chain) and E426Q-glycylthricin (B chain) are colored green, cyan, gray, magentas and yellow, respectively. The major binding-site residues and glycylthricin are well aligned without significant conformational changes.