Yoshinori Hagiwara

Induced-fitting and electrostatic potential change of PcyA upon substrate binding demonstrated by the crystal structure of the substrate-free form

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the sequential reduction of the vinyl group of the D‐ring and the A‐ring of biliverdin IXα (BV) using ferredoxin to produce phycocyanobilin, a pigment used for light‐harvesting and light‐sensing in red algae and cyanobacteria. We have determined the crystal structure of the substrate‐free form of PcyA from Synechocystis sp. PCC 6803 at 2.5 Å resolution. Structural comparison of the substrate‐free form and the PcyA–BV complex shows major changes around the entrance of the BV binding pocket; upon BV binding, two α‐helices and nearby side‐chains move to produce tight BV binding. Unexpectedly, these movements localize the positive charges around the BV binding site, which may contribute to the proper binding of ferredoxin to PcyA. In the substrate‐free form, the side‐chain of Asp105 was located at a site that would be underneath the BV A‐ring in the PcyA–BV complex and hydrogen‐bonded with His88. We propose that BV is protonated by a mechanism involving conformational changes of these two residues before reduction.

Structural Insights into Vinyl Reduction Regiospecificity of Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA)

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 IXα (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 181,182-dihydrobiliverdin IXα (18EtBV) and with biliverdin XIIIα (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.

Expression, purification and preliminary X-ray crystallographic analysis of cyanobacterial biliverdin reductase

Biliverdin reductase (BVR) catalyzes the conversion of biliverdin IX α to bilirubin IX α with concomitant oxidation of an NADH or NADPH cofactor. This enzyme also binds DNA and enhances the transcription of specific genes. Recombinant cyanobacterial BVR was overexpressed in Escherichia coli, purified and crystallized. A native data set was collected to 2.34 Å resolution on beamline BL38B1 at SPring-8. An SeMet data set was collected from a microcrystal (300×10×10 µm) on the RIKEN targeted protein beamline BL32XU and diffraction spots were obtained to 3.0 Å resolution. The native BVR crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=58.8, b=88.4, c=132.6 Å. Assuming that two molecules are present in the asymmetric unit, VM (the Matthews coefficient) was calculated to be 2.37 Å3 Da(-1) and the solvent content was estimated to be 48.1%. The structure of cyanobacterial BVR may provide insights into the mechanisms of its enzymatic and physiological functions.

Atomic‐resolution structure of the phycocyanobilin:ferredoxin oxidoreductase I86D mutant in complex with fully protonated biliverdin

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the reduction of biliverdin (BV) to produce phycocyanobilin, a linear tetrapyrrole pigment used for light harvesting and light sensing. Spectroscopic and HPLC analyses inidicate that BV bound to the I86D mutant of PcyA is fully protonated (BVH+) and can accept an electron, but I86D is unable to donate protons for the reduction; therefore, compared to the wild‐type PcyA, the I86D mutant stabilizes BVH+. To elucidate the structural basis of the I86D mutation, we determined the atomic‐resolution structure of the I86D–BVH+ complex and the protonation states of the essential residues Asp105 and Glu76 in PcyA. Our study revealed that Asp105 adopted a fixed conformation in the I86D mutant, although it had dual conformations in wild‐type PcyA which reflected the protonation states of BV. Taken together with biochemical/spectroscopic results, our analysis of the I86D–BVH+ structure supports the hypothesis that flexibility of Asp105 is essential for the catalytic activity of PcyA.

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.

Two Protonation States and Structural Features of a Bilin Reductase PcyA Revealed by Neutron Crystallography

シアノバクテリアや植物のような光合成生物は,細胞 内にビリン色素と呼ばれる光を集める色素をもっている. そのビリン色素を分子内にもつタンパク質のいくつかは, シアノバクテリアなどでは光合成,高等植物では開花や 紅葉・落葉などをコントロールするシグナル伝達の役割 を担う.フィコシアノビリン(PCB)はその両方に使われ る重要なビリン色素の1つである.PCBはヘム分解産物 であるビリベルジン(BV)から,フェレドキシンにより 供給される電子を使って酵素PcyAにより合成される.1) PcyAはまずBVのD環ビニル基を還元し,18エチルビ リベルジン(18EtBV)を生成する.その後,18EtBVのA 環を還元し,フィコシアノビリン(PCB)を合成する.そ れぞれのステップで2電子ずつ(計4電子)使い,2つず つのH+(計4つのH+)が付加される.その還元順序は変 わることなく,必ずD環ビニル基の還元がA環の還元に 先立つ(図1). 中性子結晶構造解析で明らかになったビリン還元酵素PcyA 基質複合体の2つの水素化状態と構造的特徴