title: Structural insights into vinyl reduction regiospecificity of phycocyanobilin:ferredoxin oxidoreductase (PcyA).
abstract: Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hagiwara
authors.forename: Masakazu
authors.initials: M
authors.lastname: Sugishima
authors.forename: Htoi
authors.initials: H
authors.lastname: Khawn
authors.forename: Hideki
authors.initials: H
authors.lastname: Kinoshita
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Inomata
authors.forename: Lixia
authors.initials: L
authors.lastname: Shang
authors.forename: J Clark
authors.initials: JC
authors.lastname: Lagarias
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Fukuyama
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001664:Biliverdine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052980:Phycobilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010798:Phycocyanin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D052980:Phycobilins; D010798:Phycocyanin; C011069:phycocyanobilin; D010088:Oxidoreductases; C442236:phycocyanobilin ferredoxin oxidoreductase, Synechocystis; D001664:Biliverdine