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Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome

Open AccessPublished:December 10, 2019DOI:https://doi.org/10.1016/S0021-9258(17)49934-0
      Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

      Introduction

      Phytochrome is a red light–sensitive photoreceptor in plants that is involved in photoperiodic induction of flowering, chloroplast development, leaf senescence, and leaf abscission (
      • Schafer E.
      • Bowle C.
      Phytochrome-mediated photoperception and signal transduction in higher plants.
      ). The chromophore of phytochrome for accepting red/far-red light is phytochromobilin (PΦB),
      The abbreviations used are: PΦB
      phytochromobilin
      BV
      biliverdin IXα
      15,16-DHBV
      15,16-dihydrobiliverdin IXα
      FDBR
      ferredoxin-dependent bilin reductase
      GtPEBB
      phycoerythrobilin:ferredoxin oxidoreductase from Guillardia theta
      PΦB synthase
      phytochromobilin:ferredoxin oxidoreductase
      SeMet-PΦB synthase
      selenomethionine substituted PΦB synthase
      RCCR
      red chlorophyll catabolite reductase.
      which is synthesized from a heme metabolite, biliverdin IXα (BV). PΦB synthase (EC 1.3.7.4), also known as HY2 or AUREA, which is located in chloroplasts, catalyzes the site-specific reduction of BV to produce PΦB using electrons supplied by ferredoxin (Scheme 1) (
      • Frankenberg N.
      • Mukougawa K.
      • Kohchi T.
      • Lagarias J.C.
      Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms.
      ,
      • Kohchi T.
      • Mukougawa K.
      • Frankenberg N.
      • Masuda M.
      • Yokota A.
      • Lagarias J.C.
      The Arabidopsis HY2 gene encodes phytochromobilin synthase, a ferredoxin-dependent biliverdin reductase.
      ). After the reduction, PΦB can form a covalent bond with the cysteine residue of apo-phytochrome to produce holo-phytochrome (
      • Lagarias J.C.
      • Lagarias D.M.
      Self-assembly of synthetic phytochrome holoprotein in vitro.
      ). Several PΦB synthase-deficient mutants (such as hy2 in Arabidopsis thaliana and aurea and yellow-green-2 in tomato) were characterized (
      • Terry M.J.
      • Kendrick R.E.
      The aurea and yellow-green-2 mutants of tomato are deficient in phytochrome chromophore synthesis.
      ,
      • Kendrick R.E.
      • Kerckhoffs L.H.J.
      • Pundsnes A.S.
      • Van Tuinen A.
      • Koorneef M.
      • Nagatani A.
      • Terry M.J.
      • Tretyn A.
      • Cordonnier-Pratt M.M.
      • Hauser B.
      • Pratt L.H.
      Photomorphogenic mutants of tomato.
      ,
      • Koornneef M.
      • Rolffab E.
      • Spruit C.J.P.
      Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L.) heynh.
      ). These mutants showed distinctive features such as elongated appearance with reduced anthocyanin and chlorophyll levels, which results in pale-green– or yellow-looking plants, because of the lack of mature phytochrome.
      Figure thumbnail grs1
      SCHEME 1Reaction scheme of PΦB synthase. PΦB synthase catalyzes the reduction of BV to produce 2(R), 3Z/E-PΦB using reducing equivalents from ferredoxin. The 2,3,31,32-diene system of the A-ring is site-specifically reduced. The ZZZssa configurations of BV and PΦB are displayed as observed in the present crystal structure.
      PΦB synthase is a member of the ferredoxin-dependent bilin reductase (FDBR) family. The FDBR family comprises several different but closely related proteins including phycocyanobilin:ferredoxin oxidoreductase (PcyA, EC 1.3.7.5), 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA, EC 1.3.7.2), phycoerythrobilin:ferredoxin oxidoreductase (PebB, EC 1.3.7.3), phycoerythrobilin synthase (PebS, EC 1.3.7.6), and PΦB synthase (
      • Tu S.L.
      • Rockwell N.C.
      • Lagarias J.C.
      • Fisher A.J.
      Insight into the radical mechanism of phycocyanobilin-ferredoxin oxidoreductase (PcyA) revealed by X-ray crystallography and biochemical measurements.
      ). These enzymes are widely distributed in oxygenic phototrophs. In cyanobacteria and red algae, phycobilins, which function in light harvesting for photosynthesis and in light sensing by cyanobacteriochrome, are produced from BV by FDBR. The reaction catalyzed by FDBR consists of radical formation on bilin after one-electron reduction by ferredoxin as the electron donor (
      • Tu S.L.
      • Gunn A.
      • Toney M.D.
      • Britt R.D.
      • Lagarias J.C.
      Biliverdin reduction by cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) proceeds via linear tetrapyrrole radical intermediates.
      ). The reduction sites of BV differ according to the enzyme: PΦB synthase catalyzes the reduction of the A-ring of BV, whereas PcyA catalyzes the reductions of the vinyl group of the D-ring and A-ring of BV (
      • Sugishima M.
      • Wada K.
      • Unno M.
      • Fukuyama K.
      Bilin-metabolizing enzymes: site-specific reductions catalyzed by two different type of enzymes.
      ).
      The X-ray structure of the BV–PcyA complex was the first reported tertiary structure of an FDBR (
      • Hagiwara Y.
      • Sugishima M.
      • Takahashi Y.
      • Fukuyama K.
      Crystal structure of phycocyanobilin:ferredoxin oxidoreductase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phycocyanobilin.
      ,
      • Unno M.
      • Sugishima M.
      • Wada K.
      • Fukuyama K.
      Structure–function relationships of ferredoxin-dependent bilin reductases.
      ). PcyA has a single-domain architecture and folds into an α/β/α sandwich. BV in the helical (ZZZ-all-syn or U-shaped) conformation is bound between the central β-sheet and C-terminal helices. In closed tetrapyrroles, such as heme, the configuration is limited, but open tetrapyrroles, such as BV, have a lot of cis-trans-isomers because of three methene bridges and four pyrrole rings. The ZZZ-all-syn configuration is similar to the conformation of closed tetrapyrroles. The conformation is completely different from that of bilins bound to phycobiliproteins (
      • Duerring M.
      • Schmidt G.B.
      • Huber R.
      Isolation, crystallization, crystal structure analysis and refinement of constitutive C-phycocyanin from the chromatically adapting cyanobacterium Fremyella diplosiphon at 1.66 Å resolution.
      ) and phytochromes (
      • Wagner J.R.
      • Zhang J.
      • Brunzelle J.S.
      • Vierstra R.D.
      • Forest K.T.
      High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution.
      ,
      • Yang X.
      • Kuk J.
      • Moffat K.
      Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: photoconversion and signal transduction.
      ,
      • Wagner J.R.
      • Brunzelle J.S.
      • Forest K.T.
      • Vierstra R.D.
      A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome.
      ,
      • Yang X.
      • Stojkovic E.A.
      • Kuk J.
      • Moffat K.
      Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion.
      ). The tertiary structures of the BV-bound forms of PebA (
      • Busch A.W.
      • Reijerse E.J.
      • Lubitz W.
      • Frankenberg-Dinkel N.
      • Hofmann E.
      Structural and mechanistic insight into the ferredoxin-mediated two-electron reduction of bilins.
      ) and PebS (
      • Dammeyer T.
      • Hofmann E.
      • Frankenberg-Dinkel N.
      Phycoerythrobilin synthase (PebS) of a marine virus: crystal structures of the biliverdin complex and the substrate-free form.
      ) and substrate-free PcyX (
      • Ledermann B.
      • Schwan M.
      • Sommerkamp J.A.
      • Hofmann E.
      • Béjà O.
      • Frankenberg-Dinkel N.
      Evolution and molecular mechanism of four-electron reducing ferredoxin-dependent bilin reductases from oceanic phages.
      ), which is phylogenetically closest to PcyA but catalyzes the same reaction as PebS, have also been determined. The conformation of bilin bound to these related enzymes is identical to that of bilin bound to PcyA. Red chlorophyll catabolite reductase (RCCR; EC 1.3.7.12), which is involved in chlorophyll degradation in plants, shares weak sequence similarity, similar folding properties, and substrate-binding mode with FDBRs. The substrate of RCCR is not a bilin but a compound of open tetrapyrrole. RCCR also requires reducing equivalents from ferredoxin for its enzymatic reaction (
      • Frankenberg N.
      • Mukougawa K.
      • Kohchi T.
      • Lagarias J.C.
      Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms.
      ,
      • Sugishima M.
      • Kitamori Y.
      • Noguchi M.
      • Kohchi T.
      • Fukuyama K.
      Crystal structure of red chlorophyll catabolite reductase: enlargement of the ferredoxin-dependent bilin reductase family.
      ,
      • Sugishima M.
      • Okamoto Y.
      • Noguchi M.
      • Kohchi T.
      • Tamiaki H.
      • Fukuyama K.
      Crystal structures of the substrate-bound forms of red chlorophyll catabolite reductase: implications for site-specific and stereospecific reaction.
      ,
      • Hörtensteiner S.
      • Kräutler B.
      Chlorophyll breakdown in higher plants.
      ). Tu et al. (
      • Tu S.L.
      • Chen H.C.
      • Ku L.W.
      Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis.
      ,
      • Chiu F.Y.
      • Chen Y.R.
      • Tu S.L.
      Electrostatic interaction of phytochromobilin synthase and ferredoxin for biosynthesis of phytochrome chromophore.
      ) proposed the reaction and ferredoxin interaction mechanisms of Arabidopsis PΦB synthase based on site-directed mutagenesis experiments, homology modeling, and docking simulation. However, the experimentally determined structure of PΦB synthase remains unknown.
      In this study, we constructed an overexpression system for tomato PΦB synthase (
      • Terry M.J.
      • Kendrick R.E.
      The aurea and yellow-green-2 mutants of tomato are deficient in phytochrome chromophore synthesis.
      ,
      • Muramoto T.
      • Kami C.
      • Kataoka H.
      • Iwata N.
      • Linley P.J.
      • Mukougawa K.
      • Yokota A.
      • Kohchi T.
      The tomato photomorphogenetic mutant, aurea, is deficient in phytochromobilin synthase for phytochrome chromophore biosynthesis.
      ) in Escherichia coli and determined its crystal structure in complex with BV at 1.95 Å resolution. The conformation and orientation of BV bound to PΦB synthase are completely different from those of BV bound to other FDBRs. The structures of the C-terminal additional loop and helix that are specific to PΦB synthase appear to be involved in BV binding and the interaction with ferredoxin. These features could not be estimated by homology modeling. On the basis of this structure, we propose a mechanism mediating site-specific reduction for the production of PΦB and a mechanism underlying the dissociation of the product.

      Results and discussion

      Characteristics of recombinant tomato PΦB synthase

      We examined the enzymatic activity of tomato monomeric PΦB synthase using an assay reported for Arabidopsis PΦB synthase (
      • Tu S.L.
      • Chen H.C.
      • Ku L.W.
      Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis.
      ). The Q-band of BV bound to PΦB synthase was blue-shifted following the addition of NADPH, just as in the reaction catalyzed by Arabidopsis PΦB synthase (Fig. 1), indicating formation of PΦB. Before formation of PΦB, absorption at 450 and 740 nm increased and then decreased. We did not confirm radical formation by ESR measurement, but the spectral change before the formation of PΦB was similar to that reported for Arabidopsis PΦB synthase, in which radical formation was confirmed by ESR (
      • Tu S.L.
      • Chen H.C.
      • Ku L.W.
      Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis.
      ). Thus, the increases in absorption at 450 and 740 nm suggest the formation of radical species during the reaction. HPLC analysis of the products indicated that both 3Z-PΦB and 3E-PΦB were produced as in the reaction catalyzed by Arabidopsis PΦB synthase (Fig. 1C).
      Figure thumbnail gr1
      Figure 1Enzymatic assay of tomato PΦB synthase. A, the UV-visible spectrum of BV-tomato PΦB synthase is shown as a solid line, whereas that of BV is shown as a broken line. Absorption at 640 nm increases upon binding to PΦB synthase. B, spectrum changes occurring during the conversion from BV to PΦB were monitored at 2-min intervals for 30 min. The initial and the end point spectra are shown as black bold and black dashed lines, respectively. The single-turnover reaction was initiated by the addition of NADPH. C, HPLC analysis of the product of tomato PΦB synthase. Following the single-turnover reaction shown in B, the reaction was stopped by the addition of TFA. A chromatogram obtained at 380 nm is shown. BV was purchased from Frontier Scientific.

      Overall structure of tomato PΦB synthase in complex with BV

      The crystal structure of tomato PΦB synthase in complex with BV, determined at 1.95 Å resolution, is shown in Fig. 2A. Consistent with other FDBRs and RCCRs, PΦB synthase folds into an α/β/α sandwich. The topology of the central β-sheet consists of a β-meander motif with helices H2–4 inserted between strands S6 and S7. The distal α-helical layer is composed of helices H2, H4, and H6–10, whereas the proximal α-helical layer is composed of helices H1, H3, and H5. BV is bound between the central antiparallel β sheet (S1–S7) and α-helices (H6 and H7) as in other FDBRs. Following the H8 helix, a long loop and short α-helices (H9 and H10) are observed in tomato PΦB synthase (50 amino acid residues). This loop and helical structure are specific to PΦB synthase. The recently reported structure of PebB from a cryptophyte (GtPEBB) also has a C-terminal extension (
      • Sommerkamp J.A.
      • Frankenberg-Dinkel N.
      • Hofmann E.
      Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.
      ), although the amino acid sequence and the structure of that C-terminal extension are not similar to those of PΦB synthase (Fig. 2C and Fig. S1). Sequence alignment of FDBRs showed that the amino acid sequence corresponding to this loop and H9 helix is not conserved in other FDBRs, whereas it is conserved in PΦB synthases (Fig. S1). The surface electrostatic potential demonstrated that the positive charge is localized near the entrance of the substrate-binding pocket as in other FDBRs and RCCRs, suggesting that acidic ferredoxin binds near the propionate groups of BV (Fig. 2B). The amino acid residues involved in the interactions between Arabidopsis PΦB synthase and ferredoxin 2 were identified using cross-linking and enzymatic assays (
      • Chiu F.Y.
      • Chen Y.R.
      • Tu S.L.
      Electrostatic interaction of phytochromobilin synthase and ferredoxin for biosynthesis of phytochrome chromophore.
      ). The residues corresponding to Lys-183, Arg-200, Lys-263, and Arg-264 in Arabidopsis PΦB synthase are located on the molecular surface and surrounding the entrance of BV, suggesting that they are directly involved in the interaction with ferredoxin. The Cl ion contained in the crystallization solution is located close to the propionate group of BV and the side chain of Arg-207, which corresponds to Arg-200 in Arabidopsis PΦB synthase. Upon ferredoxin binding to PΦB synthase, an acidic residue of ferredoxin may replace the Cl ion and interact with the side chain of Arg-207 and the propionate group of BV. Glu-194 and Lys-262, which correspond to Glu-187 and Lys-255 in Arabidopsis PΦB synthase, are not located on the molecular surface. These residues may be involved in the stabilization of the additional C-terminal long loop and H9 and H10 helices, which are specific to PΦB synthase and form part of the BV entrance. Thus, these two residues may be indirectly involved in the interaction with ferredoxin.
      Figure thumbnail gr2
      Figure 2Overall structure of BV-tomato PΦB synthase. A, stereo-diagram of the overall structure of tomato PΦB synthase is shown. BV is depicted as a stick model. Additional C-terminal loop and helices are shown in yellow. The N-terminal 10 residues, His tag, and C-terminal 4 residues were disordered. One Mg2+ and one Cl ions are shown as spheres. B, the surface electrostatic potential was in the range of ±5 kT/e. The potential was calculated with APBS (
      • Baker N.A.
      • Sept D.
      • Joseph S.
      • Holst M.J.
      • McCammon J.A.
      Electrostatics of nanosystems: application to microtubules and the ribosome.
      ). C, Cα trace of BVtomato PΦB synthase, superimposed onto that of the Synechocystis PcyA complex (cyan, PDB ID: 2D1E, left panel) and the 15,16-DHBVGtPEBB complex (orange, PDB ID: 6QX6, right panel). Lower panels shown as dashed boxes demonstrated the zoomed-in view of the substrates. All structural figures were prepared with PyMOL (

      DeLano, W. L., (2010) The PyMOL Molecular Graphics System, version 1.3r1, Schroedinger, LLC, New York.

      ).

      BV-binding mode in PΦB synthase

      As shown in Fig. 3A, BV bound to tomato PΦB synthase could be clearly observed. The ZZZssa conformation was observed in tomato PΦB synthase, whereas the conformation of BV bound to other FDBRs was ZZZ-all-syn (U-shaped). The conformation observed in PΦB synthase is identical to that in the Pr state of bacteriophytochrome (
      • Wagner J.R.
      • Zhang J.
      • Brunzelle J.S.
      • Vierstra R.D.
      • Forest K.T.
      High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution.
      ,
      • Yang X.
      • Kuk J.
      • Moffat K.
      Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: photoconversion and signal transduction.
      ,
      • Wagner J.R.
      • Brunzelle J.S.
      • Forest K.T.
      • Vierstra R.D.
      A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome.
      ,
      • Yang X.
      • Stojkovic E.A.
      • Kuk J.
      • Moffat K.
      Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion.
      ).
      Figure thumbnail gr3
      Figure 3BV-binding site structure of tomato PΦB synthase. A, Polder map, an improved omit map (
      • Liebschner D.
      • Afonine P.V.
      • Moriarty N.W.
      • Poon B.K.
      • Sobolev O.V.
      • Terwilliger T.C.
      • Adams P.D.
      Polder maps: improving OMIT maps by excluding bulk solvent.
      ) in which BV and catalytically important residues and water molecules are omitted and contoured by 3.0 σ, are superimposed onto the tomato PΦB synthase structure. The view from the side with the H6 and H7 helices is displayed in the left panel, and the view from the side with the A and B rings (the view in the left panel is rotated 90° anticlockwise) is displayed in the right panel. Hydrogen bonds involved in the catalysis are depicted as yellow dashed lines. B, a close-up view of the BV-binding site is shown. Residues within 4 Å from BV and water molecules involved in the reduction are shown. Hydrogen bonds involved in catalysis are depicted as yellow dashed lines. The distances between Wat-3 and the C2 atom of BV and between the side chain of Val-121 and the C21 atom of BV are shown in Å. C, comparison of the substrate-binding sites of tomato PΦB synthase (green and white [BV]) and GtPEBB (orange). Hydrogen bonds and salt bridges are depicted as dashed lines.
      One of the structural reasons why ZZZssa configuration of BV is accepted in PΦB synthase is the position of Arg-259 (Fig. 3B). If the configuration of BV bound to PΦB synthase is ZZZ-all-syn, this Arg collides with the D-ring of BV. In other words, Arg-259 acts as a dam to avoid further invasion of BV toward the interior side of the substrate-binding pocket. Also, Arg-259 interacts with the vinyl group of the D-ring of BV via a cation–π interaction to accept the ZZZssa configuration of BV. Indeed, this Arg residue is substituted by Gln in PcyA, Ser in PebA, and Met in PebS. Thus, the ZZZ-all-syn configuration of BV is accepted in other FDBRs, and BV is located on the interior side of the enzymes relative to the situation in PΦB synthase. As shown in Fig. 3 (B and C), Ser-195 forms a hydrogen bond with the lactam oxygen atom of the D-ring of BV, which is likely to stabilize the anti-conformation of the C15–C16 double bond of BV.
      Another structural reason may be the substitution of the central Asp residue. In other FDBRs, an aspartic acid residue belonging to the central strand (S5), such as Asp-105 in Synechocystis PcyA (
      • Hagiwara Y.
      • Sugishima M.
      • Takahashi Y.
      • Fukuyama K.
      Crystal structure of phycocyanobilin:ferredoxin oxidoreductase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phycocyanobilin.
      ), is located under the center of BV, and the carboxyl group of the aspartic acid side chain forms hydrogen bonds with the pyrrole nitrogen atoms of BV to stabilize the ZZZ-all-syn configuration. This structural feature is conserved in all other FDBRs, excluding 15,16-DHBV–bound GtPEBB, in which the substrate conformation is similar to that of tomato PΦB synthase (Fig. 2C). In tomato PΦB synthase, the corresponding residue is substituted by asparagine (Asn-140) and is not located under the center of BV but rather forms a hydrogen bond with the lactam oxygen atom of the A-ring of BV (Fig. 3B). Thus, this substitution may destabilize the syn-conformation of the C15–C16 double bond of BV in tomato PΦB synthase. This substitution is conserved in other higher plant PΦB synthases but not in GtPEBB (Fig. S1).
      It was proposed that this aspartic acid residue in PcyA contributes to the protonation of BV upon binding to PcyA (
      • Tu S.L.
      • Gunn A.
      • Toney M.D.
      • Britt R.D.
      • Lagarias J.C.
      Biliverdin reduction by cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) proceeds via linear tetrapyrrole radical intermediates.
      ,
      • Unno M.
      • Ishikawa-Suto K.
      • Kusaka K.
      • Tamada T.
      • Hagiwara Y.
      • Sugishima M.
      • Wada K.
      • Yamada T.
      • Tomoyori K.
      • Hosoya T.
      • Tanaka I.
      • Niimura N.
      • Kuroki R.
      • Inaka K.
      • Ishihara M.
      • et al.
      Insights into the proton transfer mechanism of a bilin reductase PcyA following neutron crystallography.
      ). The light absorption shoulder observed at 740 nm in the BV–PcyA complex, which suggests that the formation of protonated BV (BVH+), was not observed in the BV–PΦB synthase complex (Fig. 1A), suggesting that BV bound to PΦB synthase exists in the neutral form and not as BVH+. This is consistent with the structural feature of PΦB synthase, in which the central Asp is substituted with Asn.
      The lactam oxygen atom of the D-ring of BV also forms a hydrogen bond with a water molecule, which forms a hydrogen bond with Trp-258. The basic residue belonging to the C-terminal additional loop, Arg-316, forms a salt bridge with the propionate group of BV. Thr-192 forms a hydrogen bond with the propionate group of BV (Fig. 3B). Arg-207 and Lys-321 interact with the propionate group of BV via a water molecule.

      Structural comparisons with other FDBRs

      Structural comparisons with other FDBRs are shown in Fig. 2C and Fig. S2. Root-mean-square distances of Cα atoms were 1.63 Å for PcyA (116 atoms of 248 atoms were aligned), 1.21 Å for PebA (151 atoms of 244 atoms were aligned), 1.82 Å for PebS (87 atoms of 233 atoms were aligned), and 0.77 Å for GtPEBB (175 atoms of 264 atoms were aligned), respectively. BV bound to tomato PΦB synthase was flipped relative to BV bound to other FDBRs excluding GtPEBB (Fig. 2C and Fig. S2). The structure of 15,16-DHBV–bound GtPEBB, which also catalyzes the reduction of the A-ring, was recently reported (
      • Sommerkamp J.A.
      • Frankenberg-Dinkel N.
      • Hofmann E.
      Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.
      ). Indeed, the orientation of the substrate is similar to that of PΦB synthase, but the orientation of the D-ring is inverted (Figs. 2C and 3C). Phylogenetically, PebB is more closely related to PΦB synthase than to PcyA and PebA (
      • Rockwell N.C.
      • Martin S.S.
      • Li F.W.
      • Mathews S.
      • Lagarias J.C.
      The phycocyanobilin chromophore of streptophyte algal phytochromes is synthesized by HY2.
      ). Comparison of the tertiary structures of substrate-bound FDBRs also supports the idea that PΦB synthase evolved from PebB.
      Furthermore, structural comparison of PΦB synthase with PcyA, PebA, and PebS revealed that the BV-binding position in PΦB synthase is ∼3 Å close toward the entrance of the BV-binding pocket (Fig. 2C and Fig. S2). One of the reasons why the BV-binding site is slightly different from those in other FDBRs may be the substitution of the central Asp residue. As shown above, the central Asp residues, such as Asp-105 in Synechocystis PcyA, form hydrogen bonds with the pyrrole nitrogen atoms of BV to stabilize ZZZ-all-syn configuration. However, this Asp residue is substituted by Asn, and no residues form hydrogen bonds with the pyrrole nitrogen atoms of BV in PΦB synthase.
      The corresponding Asp residue in GtPEBB forms a hydrogen bond with the lactam oxygen atom of the A-ring but not with the nitrogen atom of the A-ring. Thus, the position of 15,16-DHBV is ∼1.5 Å close toward the entrance of the substrate-binding pocket (Figs. 2C and 3C). Thus, the binding position of 15,16-DHBV in GtPEBB is middle between the BV-binding position in PΦB synthase and those in other FDBRs.
      Another reason may be the existence of the C-terminal extended loop. The extended C-terminal loop interacts with the propionate groups of BV in PΦB synthase. The interaction with Arg-316 especially pulls BV toward the entrance of the BV-binding pocket relative to the substrate binding position of other FDBRs and places it in the appropriate position for A-ring reduction.
      As shown above, the substrate binding modes of PΦB synthase and GtPEBB are similar to each other, but the D-ring is in opposite orientations between GtPEBB and PΦB synthase. Ser-195, which forms a hydrogen bond with the lactam oxygen atom of the D-ring, is replaced by Ala in GtPEBB (Fig. 3C). The lactam oxygen atom of the D-ring of 15,16-DHBV bound to GtPEBB forms a hydrogen bond with Gln-114, which is substituted by Asn in PΦB synthase (Fig. 3C) (
      • Sommerkamp J.A.
      • Frankenberg-Dinkel N.
      • Hofmann E.
      Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.
      ). Thus, the D-ring is in opposite orientations between GtPEBB and PΦB synthase.

      Implications of the reaction mechanism

      Asp-263 interacts with the lactam oxygen atom of the A-ring of BV via a water molecule, Wat-1. Another water molecule, Wat-3, is located 4.2 Å from the C2 atom of the A-ring of BV (Fig. 3). Wat-3 forms a hydrogen bond with the water molecule Wat-2, and Wat-2 forms a hydrogen bond network with Asp-263 and Asp-123. These two acidic residues appear to be involved in A-ring reduction and are conserved in plant PΦB synthases (Fig. S1). To test this hypothesis, we prepared the mutated enzymes D123N and D263N, in which Asp-123 or Asp-263 are replaced by asparagine residues. Enzymatic assays showed that the D123N and D263N mutant enzymes retained BV-binding activity and radical formation activity, whereas the PΦB formation activity was negligible in the D123N or D263N mutant (Fig. 4). Similar results were reported in Arabidopsis PΦB synthase, in which mutations introduced in the corresponding acidic residues, Asp-116 and Asp-256, severely reduced the enzymatic activity (
      • Tu S.L.
      • Chen H.C.
      • Ku L.W.
      Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis.
      ). The two acidic residues and the water molecules bound to these two residues thus donate protons to the A-ring. Asp-263 functions as a first proton donor, and Asp-123 functions as a second proton donor because Asp-263 is geometrically closer to the A-ring than Asp-123, and the enzymatic activity is reduced more severely by the D263N mutation. Arg-259, of which the corresponding residue is essential for the GtPEBB reaction by forming a salt bridge with the catalytic aspartic acid residue (
      • Sommerkamp J.A.
      • Frankenberg-Dinkel N.
      • Hofmann E.
      Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.
      ), forms a salt bridge with the essential Asp-263. The water molecules, Wat-1 to Wat-3, are located between the H7 helix and BV (Fig. 3). Thus, a hydrogen atom, which is predicted to bind to the C2 atom of BV, is derived from the H7 helix side, resulting in the formation of 2(R)-PΦB. Following the formation of PΦB, the methyl group of the A-ring may move toward the central strands. The conformational change would promote the release of PΦB because of the collision between the methyl group of the A-ring of PΦB and the side chain of the conserved Val-121, which is located at a distance of 3.5 Å from the methyl group of the A-ring of BV before the reaction (Fig. 3 and Fig. S1). To confirm this hypothesis, steady-state and single-turnover analyses of V121A mutated tomato PΦB synthase were performed. Single-turnover analysis demonstrated that the V121A mutated protein was slightly slower, although it produced 3Z/E-PΦB, as well as native tomato PΦB synthase (Fig. 5, B–D), whereas no activity was detected in the V121A mutated protein in the steady-state analysis (Fig. 5E). The activity of the V121A mutated protein was not detected, even when the amount of enzyme used in the steady-state assay was increased by 2-fold. That indicates that V121A can reduce the A-ring, although the turnover rate was low. The visible spectrum of BV bound to the V121A mutated protein was similar to that of BV bound to tomato PΦB synthase, suggesting that the substrate-binding activity of the V121A mutant was normal (Fig. 5A). Therefore, the V121A mutated protein is difficult to dissociate from the product because of the lack of collision between product and enzyme.
      Figure thumbnail gr4
      Figure 4Enzymatic assay of D123N or D263N mutated tomato PΦB synthase. A, UV-visible spectra of the BV-mutated tomato PΦB synthase complex are shown as solid lines, whereas those of BV are shown as broken lines. B, spectrum changes during the conversion from BV to PΦB were monitored at 2-min intervals for 30 min. The single-turnover reaction was initiated by the addition of NADPH. C, HPLC analyses of the products of mutated tomato PΦB synthases. The single-turnover reaction shown in B was stopped by the addition of TFA. Chromatograms obtained at 380 nm are shown.
      Figure thumbnail gr5
      Figure 5Enzymatic assay of V121A mutated tomato PΦB synthase. A, the UV-visible spectrum of BV-V121A tomato PΦB synthase is shown as a solid line, whereas that of BV is shown as a broken line. B, spectrum changes during the conversion from BV to PΦB were monitored at 2-min intervals for 30 min. The initial and end-point spectra are shown as black bold and black dashed lines, respectively. The single-turnover reaction was initiated by the addition of NADPH. The conversion from BV to PΦB catalyzed by the V121A mutated protein was relatively slower than that catalyzed by native PΦB synthase, although most of the BV was converted to PΦB within 30 min. C, absorption changes at 580 nm (diamonds) and 650 nm (circles) were displayed. Decreases at 650 nm and increases at 580 nm indicate the disappearance of BV and the formation of PΦB, respectively. WT and V121A mutate protein are shown as solid and dashed lines, respectively. D, HPLC analysis of the product of the V121A mutated protein. After the single-turnover reaction shown in B, the reaction was stopped by the addition of TFA. A chromatogram obtained at 380 nm is shown. E, HPLC analysis of the product of native and V121A mutated enzymes following the steady-state reaction. The steady-state reaction was stopped by the addition of TFA. A chromatogram obtained at 380 nm is shown. In contrast to the results shown in C and D for native PΦB synthase, PΦB was not detected in the reaction with the V121A mutated protein.
      A recently discovered FDBR enzyme from streptophyte algae (KflaHY2) is phylogenetically related to PΦB synthase but can reduce the vinyl group of the D-ring and the A-ring of BV, which is similar to PcyA (
      • Rockwell N.C.
      • Martin S.S.
      • Li F.W.
      • Mathews S.
      • Lagarias J.C.
      The phycocyanobilin chromophore of streptophyte algal phytochromes is synthesized by HY2.
      ). Homology modeling of KflaHY2 based on the current tomato PΦB synthase structure (Fig. S3) suggests that the residue corresponding to Asp-263 in tomato PΦB synthase is conserved in KflaHY2, whereas the residue corresponding to Asp-123 in tomato PΦB synthase is substituted by Asn, and the residue corresponding to Asn-140 in tomato PΦB synthase is substituted by Asp, which is identical to Synechocystis PcyA. Arg-259 and Ser-195 in tomato PΦB synthase, which appear to be important for stabilizing the flipped ZZZssa conformation of BV, are also conserved in KflaHY2. Thus, the flipped ZZZssa conformation of BV is probably conserved in KflaHY2 (Fig. S3). If the BV-binding mode of KflaHY2 is similar to that of tomato PΦB synthase, Asp residues corresponding to Asn-140 and Asp-256 in tomato PΦB synthase may be involved in the enzymatic reaction. In the Synechocystis PcyA reaction, another acidic residue, Glu-76, which is adjacent to the vinyl group of the D-ring, is required for the reduction of the vinyl group of the D-ring (
      • Tu S.L.
      • Rockwell N.C.
      • Lagarias J.C.
      • Fisher A.J.
      Insight into the radical mechanism of phycocyanobilin-ferredoxin oxidoreductase (PcyA) revealed by X-ray crystallography and biochemical measurements.
      ,
      • Hagiwara Y.
      • Sugishima M.
      • Khawn H.
      • Kinoshita H.
      • Inomata K.
      • Shang L.
      • Lagarias J.C.
      • Takahashi Y.
      • Fukuyama K.
      Structural insights into vinyl reduction regiospecificity of phycocyanobilin:ferredoxin oxidoreductase (PcyA).
      ). In the homology model of KflaHY2, the acidic residue adjacent to the vinyl group of the D-ring could not be identified. Therefore, the BV-binding mode of KflaHY2 may be different from that of tomato PΦB synthase and other FDBR enzymes; alternatively, a different mechanism underlying the reduction of the vinyl group of the D-ring in KflaHY2 may be involved.
      In summary, we determined the crystal structure of tomato PΦB synthase in complex with BV at 1.95 Å resolution. The structure of the PΦB synthase-specific sequence was determined. The configuration and orientation of BV bound to PΦB synthase are completely different from those of other known BV structures bound to FDBRs. Relative to the BV-binding position in other FDBRs, BV is slightly close toward the entrance of the BV-binding pocket because of the loss of the interaction with the pyrrole nitrogens of BV and the central aspartic acid residue and the gain of interaction with Arg-316, which is specific to PΦB synthase. The present structural and enzymatic analyses suggest that two aspartic acid residues function as acidic catalysts for the reduction of the A-ring. Water molecules between the A-ring and H7 helix function as a proton relay system to donate protons. Following the reduction, the methyl group of the A-ring would collide with the side chain of Val-121 to release the product.

      Experimental procedures

      Construction of tomato PΦB synthase expression plasmids

      Amino acid sequence data for PΦB synthase from Solanum lycopersicum (tomato) (Q588D6) were retrieved from UniProt (
      • UniProt Consortium
      UniProt: a worldwide hub of protein knowledge.
      ). The nucleotide sequence corresponding to the amino acid sequence of tomato PΦB synthase (aurea), which was optimized for expression in E. coli and cloned into pUC57-Kan, was purchased from GENEWIZ (South Plainfield, NJ). Single mutation of T116S was unintentionally introduced during the plasmid design. Thr-116 is not conserved in PΦB synthases (Fig. S1) and is distant from the active site; furthermore, the resulting enzyme is active as shown in Fig. 1. To remove the chloroplast transition peptide and add a His tag at the N terminus, the ORF of tomato PΦB synthase (Met-45–Val-342) was amplified by PCR using primers 1 and 2 (Table S1). The resulting amplified fragment was fused into linearized pET-15b (Novagen) using the In-Fusion HD cloning kit (Takara Bio), creating pET-15b-aurea. The sequence of the ORF region of pET-15b-aurea was verified.
      Site-directed mutagenesis of tomato PΦB synthase was performed using the KOD Plus mutagenesis kit (Toyobo) and plasmid pET-15b-aurea as the template. The oligonucleotides shown in Table S1 were used to introduce mutations. Sequence analysis verified that the construct was free of errors.

      Expression and purification of tomato PΦB synthase

      E. coli BL21(DE3) (Novagen) was transformed with pET-15b-aurea. The transformant was grown in TB medium with ampicillin (100 μg/ml) at 28 °C. After induction with 500 μm isopropyl-β-d-thiogalactopyranoside, the cells were grown for 16 h, harvested by centrifugation, and stored at −30 °C. The following steps were performed at 4 °C or on ice. Frozen cells were thawed and suspended in 50 ml of lysis buffer (150 mm KCl and 20 mm Tris-HCl, pH 8.0) and sonicated. The membrane fraction was removed by centrifugation at 27,000 × g for 30 min. The supernatant was loaded onto a Ni-NTA-agarose column (FUJIFILM Wako Pure Chemical Corp.) equilibrated with 150 mm KCl and 20 mm Tris-HCl, pH 7.4. The column was washed with the same buffer, and the protein was eluted with an increasing linear gradient of 0 to 200 mm imidazole. Fractions containing PΦB synthase were collected and loaded onto a Hitrap Q HP column (GE Healthcare) equilibrated with 20 mm Tris-HCl, pH 7.4. The column was washed with the same buffer, and the protein was eluted with an increasing linear gradient of 0–500 mm KCl. Fractions containing PΦB synthase were concentrated, loaded onto a HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare) equilibrated with 150 mm KCl in 20 mm Tris-HCl (pH 7.4), and eluted with the same buffer. Chromatographic analysis was performed using the ÄKTA prime plus system (GE Healthcare). Fractions containing PΦB synthase were monitored by SDS-PAGE. During purification, PΦB synthase separated into two peaks in anion-exchange chromatography. Size-exclusion chromatography showed that the PΦB synthase monomer was contained in the major peak of anion-exchange chromatography, and the PΦB synthase homodimer was contained in the minor peak of anion-exchange chromatography. Because of the quantity and purity of the fraction, the monomer fraction was collected and used for further biochemical analyses including crystallization. Purified PΦB synthase was concentrated to 30 mg/ml using Amicon Ultra (Merck-Millipore) for crystallization. Selenomethionine-substituted PΦB synthase (SeMet-PΦB synthase) was expressed in E. coli B834(DE3) (Novagen), which was transformed with pET15b-aurea. Overnight Express autoinduction system 2 (Merck–Millipore) supplemented with 0.125 mg/ml l-selenomethionine was used for expression of SeMet-PΦB synthase. SeMet-PΦB synthase was purified and concentrated as described for native PΦB synthase. Typical yields of PΦB synthase and SeMet-PΦB synthase were 30 mg from 1-liter culture medium. The D123N, D256N, or V121A mutant proteins were expressed and purified as described for native PΦB synthase.

      Enzymatic assay

      Single-turnover assays of PΦB synthase activity were performed as described by Tu et al. (
      • Tu S.L.
      • Chen H.C.
      • Ku L.W.
      Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis.
      ) with the following modifications: The assay solution was prepared in an anaerobic chamber and contained 10 μm PΦB synthase or mutated proteins, 5 μm ferredoxin, 15 nm ferredoxin:NADPH oxidoreductase, and 10 μm BV in 25 mm TES-KOH buffer (pH 8.0). The reaction was initiated by adding 100 μm NADPH (final concentration) to this solution in a capped quartz cuvette on a laboratory bench. The reaction mixture was incubated at 293 K in a capped cuvette, and absorption spectra were monitored every 2 min using a SHIMADZU UV-2550 spectrophotometer. After 30 min, TFA was immediately added to the samples to stop the reaction. Steady-state assays of PΦB synthase activity were also performed as described by Tu et al. with the following modifications: the assay solution was prepared in an anaerobic chamber and contained 0.1 μm PΦB synthase or mutated proteins, 5 μm ferredoxin, 15 nm ferredoxin:NADPH oxidoreductase, and 10 μm BV in 25 mm TES-KOH buffer (pH 8.0). The reaction was initiated by adding 100 μm NADPH (final concentration) to this solution. The reaction mixture was incubated at 303 K for 30 min, and the reaction was terminated by addition of TFA.
      The reaction mixtures resulting from single-turnover and steady-state analyses were analyzed by HPLC. The samples were pretreated with Sep-Pak C18 (Millipore) and concentrated. Crude bilins were diluted using 150 μl of mobile phase solution (50% (v/v) of acetone/10 mm formic acid in water). A volume of 100 μl of diluted crude bilins was applied onto a HPLC column (InertSustainSwift C18, GL Sciences) connected to the Alliance HPLC system (Waters).

      Crystallization of the BV–PΦB synthase complex

      The BV–PΦB synthase complex was prepared by equimolar incubation of PΦB synthase and BV for 1 h on ice. The crystallization conditions for the BV–PΦB synthase complex were screened by the sitting-drop vapor-diffusion method using Mosquito at 277 K. Tiny needle-shaped crystals of the BV–PΦB synthase complex were obtained with a reservoir solution containing 20% (w/v) PEG 8000, 0.1 m Tris-HCl, pH 8.5, and 0.2 m MgCl2. Isomorphous crystals were also obtained with a reservoir solution containing 30% (w/v) PEG 4000, 0.1 m Tris-HCl, pH 8.5, and 0.2 m MgCl2. The BV–SeMet–PΦB synthase complex was crystallized using the same method.

      Data collection and structure determination

      BV–PΦB synthase and BV–SeMet–PΦB synthase crystals were soaked in crystallization solution containing glycerol up to 20% (v/v) as a cryo-protectant and frozen in liquid nitrogen. Diffraction data of the native crystal were collected at 100 K using Synchrotron radiation (λ = 1.0000 Å) from the BL32XU Beamline at the SPring-8 and EIGER X 9M hybrid photon counting detector (Dectris). A 10 μm × 10 μm microfocused X-ray beam was used to obtain diffraction data from 10–15 μm width crystals. Diffraction data for the BV–SeMet–PΦB synthase crystal were collected at 100 K using Synchrotron radiation (λ = 0.9750 Å) from the BL32XU Beamline at SPring-8. Diffraction data from 50 crystals were merged for BV–PΦB synthase to improve data quality and resolution, whereas diffraction data from a single BV–SeMet–PΦB synthase crystal was used for phase determination to reduce systematic error caused by nonisomorphism between crystals. All diffraction data were processed, merged, and scaled with KAMO (
      • Yamashita K.
      • Hirata K.
      • Yamamoto M.
      KAMO: towards automated data processing for microcrystals.
      ). Crystallographic statistics are summarized in Table 1. Rmerge and the redundancy of the native data set were relatively high because diffraction data from many crystals were merged.
      Table 1Crystal structure statistics for data collection and structure refinement
      Crystallographic data setNative PΦB synthaseSeMet PΦB synthase
      Wavelength (Å)1.00000.9750
      Space groupP43212P43212
      Unit cell (a, c; Å)64.13, 158.4563.93, 158.43
      Diffraction statistics
      The values in parentheses correspond to the highest-resolution shell.
      Maximum resolution (Å)1.95 (2.06–1.95)2.2 (2.33–2.20)
      Redundancy95.0 (91.3)6.97 (6.80)
      Completeness (%)100 (100)99.8 (98.9)
      Mean Iσ/σ(I)17.6 (3.84)13.21 (2.88)
      Rmerge (%)
      Rmerge = ∑hkl∑i|Ii(hkl) − <I(hkl)>|/∑hkl∑iIi(hkl).
      53.4 (422.2)11.0 (56.4)
      Rpim (%)
      Rpim = ∑hkl[n/(n − 1)]1/2∑i|Ii(hkl) − <I(hkl)>|/∑hkl∑iIi(hkl), where n is redundancy of the data.
      5.5 (44.7)3.5 (17.1)
      CC½0.997 (0.959)0.997 (0.864)
      Phasing
      Selenium sites7
      Figure of merit0.304
      Refinement statistics
      R factor (%)
      R factor = ∑hkl||Fo(hkl)| − |Fc(hkl)||/∑hkl|Fo(hkl)|.
      16.217.8
      Rfree (%)
      Rfree is the R factor calculated for 5% of the data not included in the refinement.
      20.721.2
      Number of atoms
      Protein23072265
      Ligands4546
      water246176
      Average B-factors (Å2)
      Protein32.5132.50
      Ligands27.7826.80
      Water40.0937.26
      Root-mean-square deviation from ideal values
      Bond lengths (Å)0.0070.004
      Bond angles (°)0.8350.641
      Ramachandran plot
      Preferred (%)97.1696.42
      Allowed (%)2.843.58
      PDB code6KME6KMD
      a The values in parentheses correspond to the highest-resolution shell.
      b Rmerge = ∑hkli|Ii(hkl) − <I(hkl)>|/∑hkliIi(hkl).
      C Rpim = ∑hkl[n/(n − 1)]1/2i|Ii(hkl) − <I(hkl)>|/∑hkliIi(hkl), where n is redundancy of the data.
      d R factor = ∑hkl||Fo(hkl)| − |Fc(hkl)||/∑hkl|Fo(hkl)|.
      e Rfree is the R factor calculated for 5% of the data not included in the refinement.
      Phase determination and auto-model building of BV–SeMet–PΦB synthase were performed using Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.-W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). The resulting automatically built model was improved manually with COOT including the addition of BV, magnesium, and chloride ions, water molecules, and several multiple conformers. The structure of native PΦB synthase was determined by Fourier synthesis using BV–SeMet–PΦB synthase. Both models were further refined and manually adjusted using Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.-W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ) and COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of COOT.
      ). Refinement statistics are summarized in Table 1.

      Author contributions

      M. S. and K. F. conceptualization; M. S. and K. W. data curation; M. S. and K. W. formal analysis; M. S., K. W., and K. Y. funding acquisition; M. S. and K. F. validation; M. S. and K. W. investigation; M. S. visualization; M. S. and K. W. methodology; M. S. writing-original draft; M. S. project administration; M. S., K. W., K. F., and K. Y. writing-review and editing; K. W. and K. Y. resources; K. F. and K. Y. supervision.

      Acknowledgments

      We thank Dr. Kunio Hirata at Riken and the staff of Beamline BL32XU at SPring-8 for assistance with data collection. We also thank Prof. Takayuki Kochi of Kyoto University for helpful discussions and construction of expression plasmids.

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