letters Structure of human biliverdin IXβ reductase, an early fetal bilirubin IXβ producing enzyme Pedro José Barbosa Pereira 1,2 , Sandra Macedo-Ribeiro 1,2 , Antonio Párraga 2 , Rosa Pérez-Luque 2 , Orla Cunningham 3 , Kevin Darcy 3 , Timothy J. Mantle 3 and Miquel Coll 2 1 These authors contributed equally to this work and share first authorship. 2 Instituto de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi Girona, 18-26, 08034 Barcelona, Spain. 3 Department of Biochemistry, Trinity College, Dublin 2, Ireland. Biliverdin IXβ reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXβ, the major heme catabolite during early fetal development. BVR-B displays a preference for biliverdin isomers without propio- nates straddling the C10 position, in contrast to biliverdin IXα reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP + at 1.15 Å resolution. Human BVR-B is a monomer displaying an α/β dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IVα, biliverdin IXα, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates nature structural biology • volume 8 number 3 • march 2001 215 and inhibitors bind primarily through hydrophobic interac- tions, explaining its broad specificity. The reducible atom of both biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the biliverdin IXα isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzyme’s preference for NADP(H) and its B-face stereospecificity. The first identifiable heme catabolite in the human fetus is bilirubin IXβ. It is detectable at 14–15 weeks 1 and remains the major bile pigment at 20 weeks 2 . Unlike bilirubin IXα, the IXβ isomer of bilirubin does not undergo internal hydrogen bonding, having a much higher solubility. Due to this property, it can be excreted without previous conjugation to glucuronic acid, a reac- tion catalyzed by UDP-glucuronyltransferase, which is not expressed at significant levels until the first week after birth. Bilirubin IXβ is transported out of the fetal liver and into bile and the lumen of the fetal intestine 1 , thereby facilitating fetal clearance of potentially neurotoxic tetrapyrroles. Heme cleavage at the β- meso position produces biliverdin IXβ, which is reduced by biliverdin IXβ reductase (BVR-B) to form bilirubin IXβ 3 . This heme catabolism pathway in utero is distinct from that in adults, in which heme cleavage occurs almost exclusively at the α- meso position, producing biliverdin IXα and bilirubin IXα. The physiological relevance of the apparent switch in heme degrada- tion from a IXβ pathway in utero to a IXα pathway at parturition may be coupled to the switch from embryonic through fetal to adult hemoglobin. It was shown that BVR-B is identical to flavin reductase (FR) 4 , which is abundant in adult erythrocytes. FR can provide free reduced flavins for the reduction of methemoglobin 5 . BVR-B is thus a promiscuous enzyme that catalyzes the NAD(P)H-dependent reduction of a range of non-α isomers of Fig. 1 BVR-B displays an α/β dinucleotide binding fold. a, Sequence alignment of human (SwissProt entry FLRE_HUMAN 28 ) and bovine (Sw issProt ent ry FLRE_BOVIN 29 ) BVR-B and related enzymes from turkey (SwissProt entry Q91089 (ref. 11)) and chicken (SwissProt entry Q90940 (ref. 11)). Amino acid residues identical to human BVR-B are shaded. Numbers above the alignment refer to the human BVR-B sequence. The secondary structure elements shown correspond to the human BVR-B structure. α-Helices are shown in red, 3 10 -helices in orange and β-strands in blue. b, Overall structure of human BVR-B in complex with NADP + . The secondary structure elements are displayed in red (α-helices) and blue (β-sheets). Important loops, and the C-terminus and N-terminus are labeled. The NADP molecule is shown as a ball-and-stick model with car- bon atoms in yellow, oxygens in red, nitrogens in blue and phosphorous in orange. a b © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com