title: Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8.
abstract: Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.
authors: Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro, Tokushima 770-8514, Japan; RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors: Takahito
authors: T
authors: Imagawa
authors: Department of Protein Structural Biology, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto 603-8555, Japan.
authors: Toshiharu
authors: T
authors: Tsurumura
authors: Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro, Tokushima 770-8514, Japan.
authors: Yasushi
authors: Y
authors: Sugimoto
authors: Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro, Tokushima 770-8514, Japan.
authors: Kenji
authors: K
authors: Aki
authors: Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro, Tokushima 770-8514, Japan.
authors: Kazumi
authors: K
authors: Ishidoh
authors: RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors: Seiki
authors: S
authors: Kuramitsu
authors: Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro, Tokushima 770-8514, Japan; RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan. Electronic address: tsuge@cc.kyoto-su.ac.jp.
authors: Hideaki
authors: H
authors: Tsuge
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000378:metabolism*
chemical_list: D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D010088:Oxidoreductases