A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity.
Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin.
Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki 889-1692, Japan.|Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki 889-1692, Japan.
Haruna
H
Takao
Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki 889-1692, Japan.
Kei
K
Hirabayashi
Advanced Analysis Center, National Agriculture and Food Research Organization, Ibaraki 305-8602, Japan.
Yuki
Y
Nishigaya
Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki 889-1692, Japan.
Haruna
H
Kouriki
Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Tetsuko
T
Nakaniwa
Department of Biochemistry and Applied Chemistry, National Institute of Technology, Kurume College, Fukuoka 830-8555, Japan.
Yoshinori
Y
Hagiwara
Department of Medical Biochemistry, Kurume University School of Medicine, Fukuoka 830-0011, Japan.
Jiro
J
Harada
Department of Medical Biochemistry, Kurume University School of Medicine, Fukuoka 830-0011, Japan.
Hideaki
H
Sato
Advanced Analysis Center, National Agriculture and Food Research Organization, Ibaraki 305-8602, Japan.
Toshimasa
T
Yamazaki
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan.
Yoichi
Y
Sakakibara
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan.
Masahito
M
Suiko
Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan.
Yujiro
Y
Asada
Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
Yasuhiro
Y
Takahashi
Department of Medical Biochemistry, Kurume University School of Medicine, Fukuoka 830-0011, Japan.
Ken
0000-0002-1531-9685
K
Yamamoto
Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.|Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
Keiichi
K
Fukuyama
Department of Medical Biochemistry, Kurume University School of Medicine, Fukuoka 830-0011, Japan.
Masakazu
0000-0002-0840-4929
M
Sugishima
Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki 889-1692, Japan.
Kei
K
Wada
D001120:Arginine / Q000737:chemistry; D001663:Bilirubin / Q000378:metabolism; D001664:Biliverdine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003067:Coenzymes; D009249:NADP; D001120:Arginine; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D001664:Biliverdine; D001663:Bilirubin