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

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.

Phytochrome is a red light-sensitive photoreceptor in plants that is involved in photoperiodic induction of flowering, chloroplast development, leaf senescence, and leaf abscission (1).
The chromophore of phytochrome for accepting red/far-red light is phytochromobilin (P⌽B), 2 which is synthesized from a heme metabolite, biliverdin IX␣ (BV). P⌽B 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 P⌽B using electrons supplied by ferredoxin (Scheme 1) (2,3). After the reduction, P⌽B can form a covalent bond with the cysteine residue of apo-phytochrome to produce holo-phytochrome (4). Several P⌽B synthase-deficient mutants (such as hy2 in Arabidopsis thaliana and aurea and yellow-green-2 in tomato) were characterized (5)(6)(7). These mutants showed distinctive features such as elongated appearance with reduced anthocyanin and chlorophyll levels, which results in palegreen-or yellow-looking plants, because of the lack of mature phytochrome.
P⌽B 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 P⌽B synthase (8). 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 (9). The reduction sites of BV differ according to the enzyme: P⌽B 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 (10).
The X-ray structure of the BV-PcyA complex was the first reported tertiary structure of an FDBR (11,12). PcyA has a single-domain architecture and folds into an ␣/␤/␣ sandwich. BV in the helical (ZZZ-all-syn or U-shaped) conformation is bound between the central ␤-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 (13) and phytochromes (14 -17). The tertiary structures of the BV-bound forms of PebA (18) and PebS (19) and substrate-free PcyX (20), 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 (2,(21)(22)(23). Tu et al. (24,25) proposed the reaction and ferredoxin interaction mechanisms of Arabidopsis P⌽B synthase based on site-directed mutagenesis experiments, homology modeling, and docking simulation. However, the experimentally determined structure of P⌽B synthase remains unknown.
In this study, we constructed an overexpression system for tomato P⌽B synthase (5,26) 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 P⌽B 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 P⌽B 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 P⌽B and a mechanism underlying the dissociation of the product.

Characteristics of recombinant tomato P⌽B synthase
We examined the enzymatic activity of tomato monomeric P⌽B synthase using an assay reported for Arabidopsis P⌽B synthase (24). The Q-band of BV bound to P⌽B synthase was blue-shifted following the addition of NADPH, just as in the reaction catalyzed by Arabidopsis P⌽B synthase (Fig. 1), indicating formation of P⌽B. Before formation of P⌽B, 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 P⌽B was similar to that reported for Arabidopsis P⌽B synthase, in which radical formation was confirmed by ESR (24). 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-P⌽B and 3E-P⌽B were produced as in the reaction catalyzed by Arabidopsis P⌽B synthase (Fig. 1C).

Overall structure of tomato P⌽B synthase in complex with BV
The crystal structure of tomato P⌽B synthase in complex with BV, determined at 1.95 Å resolution, is shown in Fig. 2A. Consistent with other FDBRs and RCCRs, P⌽B synthase folds into an ␣/␤/␣ sandwich. The topology of the central ␤-sheet consists of a ␤-meander motif with helices H2-4 inserted between strands S6 and S7. The distal ␣-helical layer is composed of helices H2, H4, and H6 -10, whereas the proximal ␣-helical layer is composed of helices H1, H3, and H5. BV is bound between the central antiparallel ␤ sheet (S1-S7) and ␣-helices (H6 and H7) as in other FDBRs. Following the H8 helix, a long loop and short ␣-helices (H9 and H10) are observed in tomato P⌽B synthase (50 amino acid residues). This loop and helical structure are specific to P⌽B synthase. The recently reported structure of PebB from a cryptophyte (GtPEBB) also has a C-terminal extension (27), although the amino acid sequence and the structure of that C-terminal extension are not similar to those of P⌽B 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 P⌽B 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 P⌽B synthase and ferredoxin 2 were identified using cross-linking and enzymatic assays (25).
The residues corresponding to Lys-183, Arg-200, Lys-263, and Arg-264 in Arabidopsis P⌽B 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 Ϫ 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 P⌽B SCHEME 1. Reaction scheme of P⌽B synthase. P⌽B synthase catalyzes the reduction of BV to produce 2(R), 3Z/E-P⌽B using reducing equivalents from ferredoxin. The 2,3,3 1 ,3 2 -diene system of the A-ring is site-specifically reduced. The ZZZssa configurations of BV and P⌽B are displayed as observed in the present crystal structure.

Structure of phytochromobilin synthase bound to substrate BV-binding mode in P⌽B synthase
As shown in Fig. 3A, BV bound to tomato P⌽B synthase could be clearly observed. The ZZZssa conformation was observed in tomato P⌽B synthase, whereas the conformation of BV bound to other FDBRs was ZZZ-all-syn (U-shaped). The conformation observed in P⌽B synthase is identical to that in the Pr state of bacteriophytochrome (14 -17).
One of the structural reasons why ZZZssa configuration of BV is accepted in P⌽B synthase is the position of Arg-259 (Fig.  3B). If the configuration of BV bound to P⌽B synthase is ZZZall-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 cationinteraction 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 P⌽B 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 (11), 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 P⌽B synthase (Fig. 2C). In tomato P⌽B 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 P⌽B synthase. This

Structure of phytochromobilin synthase bound to substrate
substitution is conserved in other higher plant P⌽B 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 (9,29). The light absorption shoulder observed at 740 nm in the BV-PcyA complex, which suggests that the formation of protonated BV (BVH ϩ ), was not observed in the BV-P⌽B synthase complex (Fig. 1A), suggesting that BV bound to P⌽B synthase exists in the neutral form and not as BVH ϩ . This is consistent with the structural feature of P⌽B 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 C␣ 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 P⌽B 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 (27). Indeed, the orientation of the substrate is similar to that of P⌽B synthase, but the orientation of the D-ring is inverted (Figs. 2C and 3C). Phylogenetically, PebB is more closely related to P⌽B synthase than to PcyA and PebA (30). Comparison of the tertiary structures of substrate-bound FDBRs also supports the idea that P⌽B synthase evolved from PebB.
Furthermore, structural comparison of P⌽B synthase with PcyA, PebA, and PebS revealed that the BV-binding position in P⌽B synthase is ϳ3 Å close toward the entrance of the BVbinding 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 P⌽B 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 ϳ1.5 Å close toward the entrance of the substratebinding pocket (Figs. 2C and 3C). Thus, the binding position of 15,16-DHBV in GtPEBB is middle between the BV-binding position in P⌽B 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 P⌽B 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 P⌽B synthase and GtPEBB are similar to each other, but the D-ring is in opposite orientations between GtPEBB and P⌽B 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 P⌽B synthase (Fig. 3C) (27). Thus, the D-ring is in opposite orientations between GtPEBB and P⌽B 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 P⌽B 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 P⌽B formation activity was negligible in the D123N or D263N mutant (Fig. 4). Similar results were reported in Arabidopsis P⌽B synthase, in which mutations introduced in the corresponding acidic residues, Asp-116 and Asp-256, severely reduced the enzymatic activity (24). 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 (27), 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)-P⌽B. Following the formation of P⌽B, the methyl group of the A-ring may move toward the central strands. The conformational change would promote the release of P⌽B because of the collision between the methyl group of the A-ring of P⌽B 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 P⌽B synthase were performed. Singleturnover analysis demonstrated that the V121A mutated protein was slightly slower, although it produced 3Z/E-P⌽B, as well as native tomato P⌽B 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 Structure of phytochromobilin synthase bound to substrate 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 P⌽B 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.
A recently discovered FDBR enzyme from streptophyte algae (KflaHY2) is phylogenetically related to P⌽B synthase but can reduce the vinyl group of the D-ring and the A-ring of BV, which is similar to PcyA (30). Homology modeling of KflaHY2 based on the current tomato P⌽B synthase structure (Fig. S3) suggests that the residue corresponding to Asp-263 in tomato P⌽B synthase is conserved in KflaHY2, whereas the residue corresponding to Asp-123 in tomato P⌽B synthase is substituted by Asn, and the residue corresponding to Asn-140 in tomato P⌽B synthase is substituted by Asp, which is identical to Synechocystis PcyA. Arg-259 and Ser-195 in tomato P⌽B 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 P⌽B synthase, Asp residues corresponding to Asn-140 and Asp-256 in tomato P⌽B 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 (8,31). In the homology model of KflaHY2, the acidic residue adjacent to the vinyl group of the D-ring could not be identified. Therefore, the BVbinding mode of KflaHY2 may be different from that of tomato P⌽B 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 P⌽B synthase in complex with BV at 1.95 Å resolution. The structure of the P⌽B synthase-specific sequence was determined. The configuration and orientation of BV bound to P⌽B 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 P⌽B 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.

Construction of tomato P⌽B synthase expression plasmids
Amino acid sequence data for P⌽B synthase from Solanum lycopersicum (tomato) (Q588D6) were retrieved from UniProt (32). The nucleotide sequence corresponding to the amino acid sequence of tomato P⌽B 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 P⌽B 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 P⌽B 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-15baurea. The sequence of the ORF region of pET-15b-aurea was verified.
Site-directed mutagenesis of tomato P⌽B 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 P⌽B synthase
E. coli BL21(DE3) (Novagen) was transformed with pET-15b-aurea. The transformant was grown in TB medium with ampicillin (100 g/ml) at 28°C. After induction with 500 M isopropyl-␤-D-thiogalactopyranoside, the cells were grown for 16 h, harvested by centrifugation, and stored at Ϫ30°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 ϫ 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 P⌽B 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 P⌽B 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 P⌽B synthase were monitored by SDS-PAGE. During purification, P⌽B synthase separated into two peaks in anion-

Structure of phytochromobilin synthase bound to substrate
exchange chromatography. Size-exclusion chromatography showed that the P⌽B synthase monomer was contained in the major peak of anion-exchange chromatography, and the P⌽B synthase homodimer was contained in the minor peak of anionexchange chromatography. Because of the quantity and purity of the fraction, the monomer fraction was collected and used for further biochemical analyses including crystallization. Purified P⌽B synthase was concentrated to 30 mg/ml using Amicon Ultra (Merck-Millipore) for crystallization. Selenomethioninesubstituted P⌽B synthase (SeMet-P⌽B 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-P⌽B synthase. SeMet-P⌽B synthase was purified and concentrated as described for native P⌽B synthase. Typical yields of P⌽B synthase and SeMet-P⌽B synthase were 30 mg from 1-liter culture medium. The D123N, D256N, or V121A mutant proteins were expressed and purified as described for native P⌽B synthase.

Enzymatic assay
Single-turnover assays of P⌽B synthase activity were performed as described by Tu et al. (24) with the following modifications: The assay solution was prepared in an anaerobic chamber and contained 10 M P⌽B synthase or mutated proteins, 5 M ferredoxin, 15 nM ferredoxin:NADPH oxidoreductase, and 10 M BV in 25 mM TES-KOH buffer (pH 8.0). The reaction was initiated by adding 100 M 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 P⌽B 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 M P⌽B synthase or mutated proteins, 5 M ferredoxin, 15 nM ferredoxin:NA-DPH oxidoreductase, and 10 M BV in 25 mM TES-KOH buffer (pH 8.0). The reaction was initiated by adding 100 M 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 l of mobile phase solution (50% (v/v) of acetone/10 mM formic acid in water). A volume of 100 l 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-P⌽B synthase complex
The BV-P⌽B synthase complex was prepared by equimolar incubation of P⌽B synthase and BV for 1 h on ice. The crystallization conditions for the BV-P⌽B synthase complex were screened by the sitting-drop vapor-diffusion method using Mosquito at 277 K. Tiny needle-shaped crystals of the BV-P⌽B 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 MgCl 2 . 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 MgCl 2 . The BV-SeMet-P⌽B synthase complex was crystallized using the same method.

Data collection and structure determination
BV-P⌽B synthase and BV-SeMet-P⌽B 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 ( ϭ 1.0000 Å) from the BL32XU Beamline at the SPring-8 and EIGER X 9M hybrid photon counting detector (Dectris). A 10 m ϫ 10 m microfocused X-ray beam was used to obtain diffraction data from 10 -15 m width crystals. Diffraction data for the BV-SeMet-P⌽B synthase crystal were collected at 100 K using Synchrotron radiation ( ϭ 0.9750 Å) from the BL32XU Beamline at SPring-8. Diffraction data from 50 crystals were merged for BV-P⌽B synthase to improve data quality and resolution, whereas diffraction data from a single BV-SeMet-P⌽B 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 (33). Crystallographic statistics are summarized in Table 1. R merge and the redundancy of the native data set were relatively high because diffraction data from many crystals were merged.
Phase determination and auto-model building of BV-SeMet-P⌽B synthase were performed using Phenix (34). 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 P⌽B synthase was determined by Fourier synthesis using BV-SeMet-P⌽B synthase. Both models were further refined and manually adjusted using Phenix (34) and COOT (35). Refinement statistics are summarized in Table 1. Figure 5. Enzymatic assay of V121A mutated tomato P⌽B synthase. A, the UV-visible spectrum of BV-V121A tomato P⌽B 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 P⌽B 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 P⌽B catalyzed by the V121A mutated protein was relatively slower than that catalyzed by native P⌽B synthase, although most of the BV was converted to P⌽B 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 P⌽B, 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 P⌽B synthase, P⌽B was not detected in the reaction with the V121A mutated protein.