PHYCOERYTHROBILIN SYNTHASE (PebS) OF A MARINE VIRUS CRYSTAL STRUCTURES OF THE BILIVERDIN-COMPLEX AND THE SUBSTRATE

FREE FORM

Thorben Dammeyer*‡, Eckhard Hofmann† and Nicole Frankenberg-Dinkel*

* From Physiology of Microorganisms and † Protein Crystallography, Biophysics, Department of Biology and Biotechnology, Ruhr-University Bochum, Bochum, Germany

‡ present address: Department of Environmental Microbiology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany

Running head: PebS X-ray structure

Address correspondence to: Nicole Frankenberg-Dinkel, Physiology of Microorganisms, Ruhr-University Bochum, Universitätsstr. 150, D-44780 Bochum. FAX:.+49 234 3214620; e-mail: nicole.frankenberg@rub.de Eckhard Hofmann, Protein Crystallography, Ruhr-University Bochum, Universitätsstr. 150, D-44780 Bochum. FAX:+49 234 3214626; e-mail: eckhard.hofmann@bph.rub.de The reddish-purple open chain tetrapyrrole pigment phycoerythrobilin (PEB) (Aλmax ~550 nm) is an essential chromophore of the light harvesting phycobiliproteins of most cyanobacteria, red algae and cryptomonads. The enzyme phycoerythrobilin synthase (PebS) recently discovered in a marine virus infecting oceanic cyanobacteria of the genus Prochlorococcus (cyanophage P-SSM2) is a new member of the ferredoxin-dependent bilin reductase (FDBR) family. In a formal four-electron reduction the substrate biliverdin IXα is reduced to yield 3Z-PEB - a reaction that commonly requires the action of two individual FDBRs. The first reaction catalyzed by PebS is the reduction of the 15,16 methine-bridge of the biliverdin IXα tetrapyrrole system. This reaction is exclusive to phycoerythrobilin biosynthetic enzymes. The second reduction site is the A-ring 2,3,31,32-diene system, the most common target of FDBRs. Here we present the first crystal structures of a PEB biosynthetic enzyme. Structures of the substrate

complex were solved at 1.8 Å and 2.1 Å resolution and of the substrate free form at 1.55 Å resolution. The overall folding revealed an α/β/α sandwich with similarity to the structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA). The substrate binding site is located between the central β-sheet and C-terminal α-helices. Eight refined molecules with bound substrate, from two different crystal forms revealed a high flexibility of the substrate binding pocket. The substrate was found to be either in a planar porphyrin-like conformation or in a helical conformation and is coordinated by a conserved aspartate/asparagine pair from the β-sheet side. From the α-helix side a conserved highly flexible aspartate/proline pair is involved in substrate binding and presumably catalysis. Phycoerythrobilin (PEB)1 is a reddish-purple pigment belonging to the group of heme-derived open-chain tetrapyrrole molecules called bilins. It functions as chromophor of light harvesting phycobiliproteins of cyanobacteria, red

http://www.jbc.org/cgi/doi/10.1074/jbc.M803765200The latest version is at JBC Papers in Press. Published on July 28, 2008 as Manuscript M803765200

Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

algae and cryptomonads. Covalently linked to phycoerythrins (PE), and some phycocyanins PEB is an essential component of many phycobilisomes (1-3). Besides this natural function phycobiliproteins are also commonly used as fluorescent probes for biotechnological and biomedical applications (4,5). The biosynthesis of all bilins starts with the common precursor molecule biliverdin IXα (BV) which is the product of oxidative cleavage of heme by the enzyme heme oxygenase (6). BV is then further reduced by highly specific ferredoxin-dependent bilin reductases (FDBRs) (7). This new family of radical enzymes, characterized by the lack of organic or metal cofactors (8,9), is comprised of several members, each targeting specific double bonds in the tetrapyrrole system. Within the FDBR family one can distinguish between two-electron reductases and four-electron reductases. Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is a four-electron FDBR and the best characterized member of this class of enzymes (8-13). It converts its substrate BV via the semireduced intermediate 181, 182-dihydrobiliverin (DHBV) to phycocyanobilin (PCB) (8). The required four electrons for the reduction are provided by the reduced form of the small, acidic redox protein ferredoxin (Fd) (Fig. 1). In vivo the electrons for Fd reduction most likely originate from photoreduced photosystem I and are then passed on to a variety of Fd-dependent enzymes (14). In contrast to the biosynthesis of PCB, two independent biosynthetic pathways leading to PEB have been described (Fig. 1). In PE-containing cyanobacteria, BV is converted by two consecutive two-electron reductions to PEB (15,16). The first reduction is catalyzed by 15, 16-DHBV:ferredoxin oxidoreductase (PebA) and yields 15, 16-DHBV. The product of this first reduction then serves as a substrate for PEB:ferredoxin oxidoreductase (PebB) to generate PEB. A second pathway leading to PEB has recently been described by

discovery of a new member of the FDBR family (17). Phycoerythrobilin synthase (PebS) (EC 1.3.7.6) catalyzes the ferredoxin-dependent four-electron reduction of BV to PEB (Fig. 1). Biochemical analyses have established that the order of reduction is identical to that catalyzed by the consecutive action of PebA and PebB. The intermediate 15, 16-DHBV is thought to remain in the active site pocket of PebS and not to be released during catalysis (17). This observation is in agreement with results on PebA and PebB from Synechococcus sp. WH8020 that suggest metabolic channeling of 15, 16-DHBV directly from PebA to PebB (15). Compelling about PebS is not only its novel activity but also its widespread distribution in wild cyanophage populations but absence in the respective host populations (17). Therefore PebS is a biologically and biochemically exceptional enzyme. Here we present high resolution crystal structures of this novel enzyme, PEB synthase alone and in complex with its substrate BV. We provide further insights into the structural basis of substrate recognition and specific reduction of the tetrapyrrole system in this interesting family of radical enzymes. EXPERIMENTAL PROCEDURES Recombinant production and purification of PebS Heterologous expression of cyanophage pebS was performed in Escherichia coli BL21 (DE3) using a pGEX-6P-3 (GE Healthcare) construct for a translational fusion to glutathione-S-transferase (GST) as described before (17). The recombinant protein product resulted in nine additional N-terminal amino acids that remained after cleavage with PreScission™ protease (GE Healthcare). For production of selenomethionine labeled protein pre-cultures and expression cultures were grown in glucose supplemented (0.4%) M9- instead of Luria Bertani-media. 20 min prior to induction with 100 µM isopropyl-β-D-thiogalactopyranoside

2

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

(IPTG) the cultures were cooled down and an amino acid mixture containing 50 mg/L final concentration L(+)-selenomethionine (Acros) was added to inhibit methionine biosynthetic pathways according to (18). Cells were harvested 15 h post induction, lysed, centrifuged and purified as described before (17) with the following modifications: ultracentrifugation was carried out at 170,000 × g for 1 h and all buffers where adjusted to pH 8.0 and contained 200 mM potassium chloride and up to the final dialysis step 5 mM dithiothreitol (DTT) for selenomethionine-proteins. The purified proteins were then concentrated using a nitrogen-pressurized, stirred ultrafiltration cell with a 10.000 MW filter cut-off (Millipore). The concentrated protein was incubated with an excess of BV (Frontier Scientific, Inc.) dissolved in DMSO for 1 h on ice in the dark. To remove free BV and to reduce salt concentration the protein solution was applied to a NAP™5 desalting column (GE Healthcare) and eluted with 25 mM TES-KOH pH 8.0. The protein concentration was determined using the calculated molar extinction coefficient (ε280 nm) (19) and adjusted to the respective concentrations. The activity of the native and selenomethionine labeled proteins was assayed as described before (17). Crystallization of PebS and PebS-BV Complex Crystallization conditions were screened by the sitting drop vapor-diffusion method using the Classic, Cryo and the JSCG - Suite (Qiagen), applying 100nL:200nL and 100nL:100nL mixtures of the protein solution (10.5 mg/mL):reservoir solution incubated at 18°C in the dark. Initial native PebS-BV crystals (clusters of green thin plates) grew in a 1:1 mixture of protein solution (10.5 mg/mL) and reservoir solutions containing 1.6 M sodium-citrate. These conditions were further optimized with the hanging drop vapor diffusion method and final crystals grew within 14 days in a 10 µL drop. The drop was taken from a 1:1 mixture of protein solution (7.5 mg/mL):1.025 M

sodium-citrate containing 4% glycerol. The mixture was incubated 15 h at 18°C and was centrifuged (2 × 30 min., 14,000 rpm) before placing the drop over a reservoir solution of 1.25 M sodium-citrate at pH 8.0. Initial selenomethionine labeled SePebS-BV crystals (green) grew in a 2:1 mixture of protein solution (8.5 mg/mL):reservoir solution containing 0.2 M magnesium chloride, 0.1 M bis-Tris pH 5.5, 25% (w/v) PEG3350. Final crystals grew in 5 µL hanging drops and were harvested in cryoloops, briefly soaked in motherliquor with 20% PEG400 as cryoprotectant prior to flash freezing in liquid nitrogen. Substrate free (colorless) selenomethionine-crystals (SePebS) grew in a 1:1 mixture of protein and reservoir solution containing 0.085 M HEPES sodium salt pH 8.5, 17% (w/v) PEG4000, 8.5% isopropanol and 15% glycerol. Data Collection and Structure Determination Oscillation data were collected at 100K at the Swiss Light Source (Villigen, Switzerland) at beamlines PXI and PXII using Mar CCD-225 detectors. All data were processed and scaled using the XDS package (20). Data statistics are listed in Table 1. Dataset SePebS-1 was used for structure solution with the Shelx package (21), using the hkl2map interface (22). Six selenium atoms were correctly located by shelxd (23) (Resolution 45-2.2Å, CC (all/weak) 55.69 / 36.66, PATFOM of 31.6) and used for phasing and density modification in shelxe (45-1.7Å, Pseudo-Free-CC correct/incorrect hand 80.03/54.25). With the resulting phases 203 residues could be automatically traced with Arp/Warp 6.0 (24). The model was then iteratively refined against the high resolution dataset SePebS-2 using refmac (25) and coot version 0.1.2 (26). Model and refinement statistics are given in Table 1. Residues 1-21, 52-56 and the active site residues D206/P207 could not be modelled due to missing electron density. This structure represents the substrate free protein (pdb accession number 2VCL). The resulting

3

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

model was then used to solve the structure of SePebS-BV in space-group P1 using programs of the CCP4-package (27). Four molecules in the unit cell were found all with bound BV in the active site. The model was again improved using iterative rounds of refinement in refmac (25) and rebuilding in coot (26). Model and refinement statistics are given in Table 1. The first 20 residues and region 52-56 were not modelled due to weak or missing density (pdb accession number 2VCK). Crystals of unlabeled PebS with bound BV (PebS-BV) diffracted similarly well than the selenomethionine labeled crystals but frequently showed multiple lattices. The structure was solved by MR in Phaser (28) using chain A of SePebS-BV as search model and refined as described before. Again four molecules in the unit cell were found all with bound BV in the active site pocket. In the final model the first 20 residues (18 in the case of chain A) have been omitted due to missing density. The loop around Asp55 could be modelled in chain D, for the other chains some residues were omitted due to disorder (chain A: 53-55, chain B: 55-56, chain C: 54-55) (pdb accession number 2VGR). In all cases medium NCS restraints have been used throughout refinement and have been released for variable parts of the structure where appropriate. All structural Figures were produced using PyMOL (29) RESULTS PebS mediates the transfer of four electrons from ferredoxin to BV PebS is only the second FDBR, after PcyA, known to perform a formal four-electron reduction (17). Biochemical analyses established that the recombinant protein is most active under slightly basic conditions (~ pH 8.0). The in vitro reduction is faster employing the recombinant phage-encoded Fd (PetF_PSSM-2) for electron delivery compared to the use of Synechococcus sp. PCC7002 Fd, resulting in an estimated turnover of ~10 min-1 for BV at room temperature (data not shown). This is

likely due to more efficient electron transfer among the phage proteins. The A-ring reduction of BV results in two possible stereoisomeric ethylidene groups, 3Z and 3E. Discussions in the past have always led to the question whether the 3Z or the 3E form is the primary product of certain FDBRs or whether they occur simultaneously. In this study we confirmed 3Z-PEB as the primary product of PebS by absorbance spectroscopy (Fig. S1). However, ratios of 3Z-PEB and 3E-PEB obtained through HPLC analyses are distorted because sample preparation tends to favor conversion to the 3E-isomer (7)(and data not shown), the thermodynamically more stable isomer (30,31). The formation of the 3Z-form as the primary product is in agreement with early studies on oat phytochromobilin (PΦB) synthase and other bilin reductases (7,32,33). Structure determination X-ray data of PebS in three different crystal forms have been collected: Wild type protein was crystallized with bound substrate BV (PebS-BV) and selenomethionine labeled protein was crystallized with and without substrate (SePebS-BV and SePebS respectively). The structure of SePebS was determined by single wavelength anomalous dispersion phasing and refined at 1.55Å resolution to an R-factor of 0.174 and a free R-factor of 0.194 (Tab. 1). This structure was substrate free and subsequently used as a search model to determine the structures of the BV-complexes. Both SePebS-BV and PebS-BV crystallized in space group P1 with similar cell dimensions. In both cases four molecules were found in the asymmetric unit. The structures were refined to R-factors of 0.184/0.227 and free R-factors of 0.225/0.285 using data to 1.8 Å and 2.1 Å resolution, respectively (Tab. 1). In all three structures the N-terminal 20 residues were found to be disordered. Residues 51-56 form a flexible β-turn that was completely resolved only in chain D of PebS-BV. In the substrate free form

4

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

SePebS the active site residues Asp206/Pro207 could not be conclusively modeled. All Ramachandran-plot outliers could be assigned to residues either involved in substrate coordination or crystal contact formation. PebS is a globular single-domain protein The PebS enzyme is a single domain globular protein with dimensions of approximately 45 × 36 × 33 Å showing an α/β/α-sandwich fold (Fig. 2). A central seven stranded antiparallel β-sheet is flanked by two α-helices on one side and three α-helices on the side of the substrate binding pocket. The substrate BV is bound in a cavity formed by the β-sheet (proximal side) and by helices H3 and H4 and a long flexible loop between strands S6 and S7 (distal side) which closes in upon substrate binding. The flexibility of this region is illustrated in the Cα superposition of all nine copies of PebS present in the three crystal forms used in this study (Fig. 3). The overall fold of PebS is similar to that of cyanobacterial phycocyanobilin:ferre-doxin oxidoreductase (PcyA) from Synechocystis sp. PCC 6803 (pdb code 2D1E and 2DKE) (10,11) and Nostoc sp. PCC 7120 (pdb code 2G18) (12) (Fig. 4, Tab. S1) which belong to the same family of FDBRs. Helix H3 is shorter than the corresponding helix of PcyA and the flexible loop between strands S6 and S7 forms two short helices in PcyA that are not present in PebS (Fig. 4, 6). Both proteins bind the same substrate BV but specifically reduce it at distinct double bonds yielding to the isomers PCB (PcyA) and PEB (PebS). Substrate recognition Clear electron density for the vinyl-groups defines the orientation of the substrate with the propionate side chains facing towards the solvent (Fig. 2, 5 and Fig. S2, S3, S5). Rings A and D are buried inside the binding niche and are coordinated from the β-sheet by residues Asn88 (β-strand S4) and Asp105 (β-strand S5) (Fig. 5). Their sidechains form a hydrogen bonding network with each other and the carbonyl

oxygens of BV. These residues are comparably positioned in structure (Fig. S4A) and sequence alignment (Fig. 6) to the critical catalytic pair His88/Asp105 of PcyA (10,12,13). On the distal side, the substrate is flanked by two α-helices (H3/4) which are connected by a short linker containing an Asp/Pro pair (Asp206/Pro207) that is strictly conserved amongst FDBRs except in PcyA. Interestingly, different binding modes for the substrate BV were identified within different molecules in the crystallographic asymmetric unit (ASU) of both crystal forms (Fig. 5 and Fig. S5). In SePebS-BV three molecules show almost identical BV binding with a water molecule being in hydrogen bonding distance to all pyrrole nitrogens and to Asp206 (Fig. 5A). Here, BV is bound in a porphyrin-like planar conformation (Table S2) with the propionate side chains facing the solvent side. Within SePebS-BV the fourth molecule in the ASU shows a different binding mode of BV. Asp206 is positioned closer to the pyrrole nitrogens with one of the carboxylic oxygens replacing the pyrrole water (Fig. 5B). In the native structure PebS-BV again four different molecules are found in the ASU. In three of these the flexibility of the distal side is visible as Asp206 is rotated outside the active site (Fig. 5C). The absence of Asp206 then allows the substrate to adopt a helical lock-washer conformation with the A-ring shifted partially above ring D (Table S2). This position is visible in chains A (Fig. 5D, Fig. S3A) and C (Fig. S3C, S5C). Due to this conformational change the second site of the reduction, the C2 position of the 2,3,31,32 diene system of BV is positioned in proton accepting distance to O19. DISCUSSION FDBR enzymes catalyze the regio- and stereospecific reduction of BV to different bilin chromophores which are essential for light harvesting in phycobilisomes and light sensing in phytochromes. While the enzyme responsible for the four-electron

5

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

reduction of BV to PCB has structurally and functionally been well characterized, no structural information has been available for an enzyme involved in the synthesis of PEB. The high resolution structures of PebS from the cyanophage PSSM-2 presented in this work shed light onto the mechanisms of regiochemical control in related reductases which operate on the same substrate. At the same time it raises questions whether there is a common conserved mechanism for the reduction of the A-ring vinyl group which takes place in all FDBRs except PebA. PebS shows the same overall architecture as PcyA (Fig. 4), even though the conservation between the enzymes on the sequence level is rather low (14 % identity, 25 % similarity) (Fig. S6). However, critical residues for substrate binding are conserved but distinct differences in the active site environment seem to be the source of the chemical specificity. Initial step-Binding and protonation of BV The reaction starts with the binding of the substrate BV which is accompanied by a structural change of the protein. The most obvious structural changes in PebS occur within a flexible loop that covers the active site (Fig. 3). Upon binding, BV and PebS form a spectroscopically distinct complex (Fig. S7). This appears to be a common phenomenon for biochemical characterized members of the FDBR family (8,15)(Tu, S.-L. et al. accompanying paper; Frankenberg-Dinkel, unpublished observation). Interestingly, the spectroscopic signatures for the FDBR:BV complexes differ from each other reflecting different binding modes or protein environments in the active sites. While the long wavelength absorption of BV is blue shifted upon binding to PcyA and HY2 (8)(Tu, S.-L., accompanying paper) it is red shifted in the PebS:BV complex (Fig. S7). The PcyA:BV complex shows an additional shoulder in the near infrared (NIR) region (Fig. S7) which has been attributed to a protonated BV species as the initial step of the PcyA-reaction (12). Protonation is thought to occur via

the carbonyl-oxygen coordinating residue Asp105 (Synechocystis PcyA nomenclature). This residue (Asp105 in PebS) is highly conserved among almost all FDBRs (Fig. S6) and is involved in coordinating O1 and O19 of BV (Fig. 5). Interestingly, the BV-complex of PebA from Synechococcus sp. WH8020 shows absorption properties more similar to PebS:BV than to PcyA:BV that may indicate the structural and mechanistic relationship of both 15,16 reduction enzymes (15,17). With respect to the different binding modes of BV observed in the crystal structure it is also likely that the absorption spectrum of the PebS:BV complex rather resembles a mixture of protonated and unprotonated species than only one particular BV species. Analysis of possible H-bonding patterns within the BV conformations found in our PebS structures suggests that protonation of the carbonyl-oxygens of BV in the planar conformations is unlikely. In contrast, in the helical conformation O19 of BV is involved in a putative branched H-bond with Asp105. Consequently a protonation at this position is likely which would facilitate subsequent electron transfer. Helical conformations of BV are stable in solution but are destabilized if compensation for the intra-chromophoric hydrogen bond is provided by other optimally oriented donor groups (34). Evidence for the presence of the helical conformation in PebS:BV complexes in solution was obtained by circular dichroism spectroscopy. The spectrum revealed a strong positive signal at ~380 nm that is weaker for PcyA:BV, and a negative signal at ~680 nm (Fig. S8). These signs are characteristic for M-helical bilin conformations (35) like those in 2VGR chain A and C (Fig. 5D and Fig. S5C). In addition to the spectroscopic differences of the complexes, none of the observed binding modes of BV in PebS superposes perfectly with the situation in PcyA, reflecting the structural and functional divergence of the enzymes (Tab. S1).

6

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Based on our spectroscopic data (Figs. S7 and S8) and the helical conformation of BV found in two molecules of the ASU (2VGR) (Figs. 5D and S5C) we propose a protonation of BV as the initial step in the PebS reaction. This is achieved via Asp105 to O19 of the substrate BV in the helical conformation. In agreement with the postulated PcyA mechanism this protonation is most likely not involved in the reduction of BV but rather generates a BVH+ cation that can readily accept one or two electrons from reduced Fd. Coupled electron-proton transfer Common to all FDBRs is the direct coupling of electron and proton transfer steps. The electrons are provided by the single electron transfer protein Fd. Due to the lack of metal or organic cofactors within the FDBRs the reactions are thought to occur via substrate radical intermediates as it has previously been shown for PcyA using EPR spectroscopy (9). This has now also been proven for plant PΦB synthase (Tu, S-L, et al., accompanying paper) and can be expected to be general to FDBRs. Surface charge patterns of PcyA suggested an overlap of the Fd docking site with the substrate binding pocket (11). Hence, electron transfer could proceeds involving the propionate sidechains of BV. It is generally accepted that electrons rapidly redistribute in the tetrapyrrole ring system. While the existence of BV radicals has been shown experimentally for PcyA and HY2, the exact chemical nature of such a radical remains obscure. It can be expected that the unpaired electron is delocalized over the entire conjugated π-system. One might expect high and low spin densities at certain carbons which are likely influenced by the conformation, the binding environment and protonation. The first reduction: D-ring After initial protonation of BV and a subsequent one (or two) electron transfer to generate either a neutral or anionic radical, the first proton transfer(s) have to occur to finalize the first reduction. In contrast to PcyA which performs a regiospecific reduction at the exo-vinyl

group of BV, PebS has to transfer a proton stereospecific to C16 to generate the R configuration. Therefore, ultimately the proton must originate from the β-sheet side of the active site. Careful inspection of all structures failed to identify proton donating residues in the vicinity of the C15-C16 double bond. This is in accordance with PebA homology models based on the PcyA structure (12) and also for a provisional PebA model based on PebS (2VCK chain C, Fig. S4B). Therefore, both C15-C16 double bond reducing enzymes (PebA and PebS) might work via a similar mechanism, different from PcyA, not involving direct protonation from active site amino acid residues. The proposed reaction mechanism of PcyA is based on the concept of proton transfer involving the “critical-pair” Asp105/His88 and the carboxyl group of Glu76 alternating with direct electron transfer from Fd to the substrate. Central to this concept is a conserved proton transfer pathway leading from the solvent to the active site residues, which allows initial formation of a BVH+ cation upon substrate binding and subsequent reprotonation and delivery of additional protons. In the PebS structure no residues equivalent to Glu76 are found, and His88 is replaced by Asn88 thereby interrupting the proton relay and effectively inhibiting the D-ring vinyl reduction by the mechanism found in PcyA. One possible explanation for the PebS (and possibly PebA) mechanisms lies in the observed structural heterogeneity of the PebS-substrate complexes in our crystals. They demonstrate that conformational changes of the complex can expose the C15 and C16 carbon atoms of the C-D methine bridge temporarily to the solvent. Therefore, the two protons for the full reduction could be donated from the bulk solvent while the correct stereochemistry is enforced by the accessibility restraints imposed by the protein surface. The second reduction:

7

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

The second reduction, the reduction of the A-ring vinyl group, is the most common reaction in the FDBR family. While PebA is the sole FDBR that does not catalyze this reduction, PebS is the only enzyme that combines A-ring vinyl group reduction with a preceding 15,16 reduction in one enzyme. The corresponding A-ring vinyl reduction of 181,182-DHBV catalyzed by PcyA was proposed to proceed from the central Asp105 via His88/O1 to C2 followed by a second proton from the bulk solvent to C32 upon product release (12). In Arabidopsis thaliana HY2 His88 is replaced by Asp116 which is postulated to be functionally different (Tu, S.-L., et al. accompanying paper). Notably, in both PebB and PebS His88 is replaced by Asn88, which cannot serve as the central proton donor. One possible solution for this discrepancy lies in the helical conformation observed in our PebS-BV structure. Here, C2 is well positioned for protonation by Asp105 via O19, to yield the R-stereochemistry at C2. Interestingly, in this conformation a cavity is found adjacent to the A-ring vinyl group, in which we could locate two water molecules. This is reminiscent of the situation in PcyA where a similarly placed water molecule has been postulated to be the second proton donor (12). In HY2, A-ring reduction has been found to be critically dependent on Asp256 which is postulated to be the first proton donor (Tu, S.-L., et al., accompanying paper). This Asp residue is highly conserved in the whole FDBR family and corresponds to Asp206 in PebS. Besides its role in substrate binding (see paragraph below) we do not have any biochemical evidence for a function of this residue as a proton donor in the PebS reaction. As the carboxyl group of Asp206 is involved in a hydrogen bonding network with the pyrrole nitrogens in several observed conformations, we cannot rule out a catalytic role in PebS. Further studies on site-directed mutants of this residue will further clarify this question. Binding flexibility

One central result of this work is the demonstration of different binding modes of BV inside the binding pockets. These occur coupled to the movements of Asp206, which in turn are most likely facilitated by Pro207 since X-Pro bonds are known to enable a number of different conformations with roughly equal probability (36,37). The intrinsic flexibility of the Asp-Pro motif and the ability to respond conformationally to specific interactions may play an essential role by mediating the interconversion of the different states. However, the conformations observed in our crystal structures will not truly reflect the situation of the substrate during different stages of the reduction, as they all can be considered as ground state. Under physiological conditions the catalytically relevant binding conformations will depend critically on the radical and protonation state of the intermediates. Notably, the reaction intermediate 15,16-DHBV is reduced in the tetrapyrrole ring system and is therefore more flexible at the C-D ring bridge. In general, the PebS binding pocket seems flexible enough to allow the productive binding of all reaction intermediates in one orientation. As we observed the same orientation of BV in PebS as has been shown for PcyA (10) we expect this to be the general binding mode in all FDBRs. The same binding orientation has been published in a PebA homology model based on the PcyA structure. In this case the authors argued against this orientation since it did not place the C15-C16 double bond in close vicinity of a proton donor. Therefore, an inverted orientation of BV in PebA was postulated (12). Given our structural data we conclude that inverted substrate binding, as well as the use of a two enzyme system is not a biochemical necessity arising from the sites to be reduced upon conversion of BV to PEB (17). One should bear in mind that the reaction also involves a repetitive binding of ferredoxin, an event which should be controlled by the chemical state of the bound substrate and

8

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

9

most likely will involve a subtle conformational response in PebS upon binding. These effects will be very difficult

to assess until we are able to obtain structural information about the PebS-Fd complex.

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

REFERENCES 1. Ong, L. J., and Glazer, A. N. (1987) J. Biol. Chem. 262, 6323-6327 2. Ritter, S., Hiller, R. G., Wrench, P. M., Welte, W., and Diederichs, K. (1999) J. Struct.

Biol. 126, 86-97 3. Ong, L. J., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9515-9527 4. Glazer, A. N. (1994) J. Appl. Phycol. 6, 105-112 5. Kronick, M. N., and Grossman, P. D. (1983) Clin. Chem. 29, 1582-1586 6. Cornejo, J., Willows, R. D., and Beale, S. I. (1998) Plant J. 15, 99-107 7. Frankenberg, N., Mukougawa, K., Kohchi, T., and Lagarias, J. C. (2001) Plant Cell

13, 965-978 8. Frankenberg, N., and Lagarias, J. C. (2003) J. Biol. Chem. 278, 9219-9226 9. Tu, S. L., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004) J. Am.

Chem. Soc. 126, 8682-8693 10. Hagiwara, Y., Sugishima, M., Takahashi, Y., and Fukuyama, K. (2006) Proc. Natl.

Acad. Sci. USA 103, 27-32 11. Hagiwara, Y., Sugishima, M., Takahashi, Y., and Fukuyama, K. (2006) FEBS Lett.

580, 3823-3828 12. Tu, S. L., Rockwell, N. C., Lagarias, J. C., and Fisher, A. J. (2007) Biochem. 46,

1484-1494 13. Tu, S. L., Sughrue, W., Britt, R. D., and Lagarias, J. C. (2006) J. Biol. Chem. 281,

3127-3136 14. Blankenship, R. E. (2001) Nat. Struct. Biol. 8, 94-95 15. Dammeyer, T., and Frankenberg-Dinkel, N. (2006) J. Biol. Chem. 281, 27081-27089 16. Dammeyer, T., Michaelsen, K., and Frankenberg-Dinkel, N. (2007) FEMS Microbiol.

Lett. 271, 251-257 17. Dammeyer, T., Bagby, S. C., Sullivan, M. B., Chisholm, S. W., and Frankenberg-

Dinkel, N. (2008) Curr. Biol. 18, 442-448 18. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J.

(1993) J. Mol. Biol. 229, 105-124 19. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326 20. Kabsch, W. (1993) J. Appl. Cryst. 26, 795-800 21. Sheldrick GM, S. T. (1997) SHELXL: High Resolution Refinement, Academic Press,

Orlando, Florida 22. Pape, T., and Schneider, T. R. (2004) J. Appl. Crystall. 37, 843-844 23. Schneider, T. R., and Sheldrick, G. M. (2002) Acta Crystallogr. D Biol. Crystallogr.

58, 1772-1779 24. Morris, R. J., Perrakis, A., and Lamzin, V. S. (2002) Acta Crystallogr. Sec. D 58, 968-

975 25. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. D Biol.

Crystallogr. 53, 240-255 26. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 2126-

2132 27. Collaborative Computational Project, N. (1994) Acta Crystal. Sec. D 50, 760-763 28. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and Read, R. J. (2005) Acta

Crystallogr. D Biol. Crystallogr. 61, 458-464 29. DeLano, W. L. (2002) The PyMOL Molecular Graphics System 30. Rüdiger, W., Brandlmeier, T., Blos, I., Gossauer, A., and Weller, J.-P. (1980) Z.

Naturforsch. 35c, 763-769 31. Weller, J. P., and Gossauer, A. (1980) Chem. Ber. 113, 1603-1611.

10

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

32. McDowell, M. T., and Lagarias, J. C. (2001) Plant Physiol. 126, 1546-1554 33. Terry, M. J., McDowell, M. T., and Lagarias, J. C. (1995) J. Biol. Chem. 270, 11111-

11118 34. Krois, D. (1991) Monatsh. Chem., 495-506 35. Krois, D. L., H. (1995) Monatsh. Chem., 349-354 36. Ri, Y., Ballesteros, J. A., Abrams, C. K., Oh, S., Verselis, V. K., Weinstein, H., and

Bargiello, T. A. (1999) Biophys. J. 76, 2887-2898 37. Pastore, A., Harvey, T. S., Dempsey, C. E., and Campbell, I. D. (1989) Eur Biophys J

16, 363-367 38. Diederichs, K., and Karplus, P. A. (1997) Nat. Struct. Biol. 4, 269-275 39. Laskowski, R., and Maryanski, M. (1993) Bull. Environ. Contam. Toxicol. 50, 232-

240 FOOTNOTES * We thank Drs. Carsten Kötting, Bernhard Kräutler, Wolf-Dieter Schubert and Shih-Long Tu for helpful discussion. Thanks are also due to the beamline staff at the Swiss Light Source (Villigen, Switzerland). This work was supported by the Sonderforschungsbereich 480 of the Deutsche Forschungsgemeinschaft (to E.H. and N.F.D; Teilprojekt C6 and C8). 1 Abbreviations used are: ASU, asymmetric unit; BV, biliverdin IXα; DHBV, dihydrobiliverdin; DTT, dithiothreitol; Fd, ferredoxin; FDBR, ferredoxin-dependent bilin reductase; GST, glutathione-S-transferase; IPTG, isopropyl-β-thiogalactopyranosid; NIR, near infrared; PE, phycoerythrin; PEB, phycoerythrobilin; PebA, 15, 16-dihydrobiliverdin:ferredoxin oxidoreductase; PebB, phycoerythrobilin:ferredoxin oxidoreductase; PebS, phycoerythrobilin synthase; PΦB, phytochromobilin; PcyA, phycocyanobilin:ferredoxin oxidoreductase; RMSD, root mean square deviation SUPPORTING INFORMATION Two supplementary tables and eight supplementary figures are available online.

11

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Tables Table 1. Data Collection and Refinement Statistics a Data set SePebS1

(anomalous statistics) SePebS2 SePebS-BV PebS-BV

Beamline SLS PXII (30.6.07) SLS PXII (30.6.07) SLS PXII (30.6.07 SLS PXI (29.4.07) Resolution (Å) 45-1.7 (1.8-1.7) 38-1.55 (1.6-1.55) 49-1.8 (1.9-1.8) 48-2.1 (2.3-2.1)

Cell parameters (a,b,c; Å) (α,ß,γ; degrees)

42.50, 72.47, 90.94 90, 90, 90

42.48, 72.45, 90.94 90, 90, 90

53.56, 67.57, 81.28 92.26, 109.07, 106.58

53.99, 70.04, 81.67 75.53, 70.74, 71.13

Spacegroup P212121 P212121 P1 P1 Wavelength 0.97906 0.9826 0.97887 0.98011 Completeness (%) 100 (100) 98.8 (90.2) 97.1 (95.9) 97.5 (96.9) Multiplicity 7.7 (7.5) 26.3 (14.4) 8.1 (8.2) 4.3 (4.4) Average I/σI 16.9 (4.8) 34.1 (4.9) 13.2 (5.0) 12.8 (5.3) Rsym (%) 8.9 (56.5) 7.6 (64.1) 10.5 (42.0) 8.0 (29.7) Rmeas (%)b 9.5 (60.7) 7.7 (66.5) 11.2 (44.9) 9.1 (33.9) Rmrgd-F (%)b 8.2 (34.6) 4.8 (31.7) 7.6 (26.7) 9.0 (25.3)

Phasing Shelxd Resolution for phasing (Å) 45-2.2

Number of scatteres found 6 CC (All/weak) 55.69 / 36.66 PATFOM 31.60 Shelxe resolution range (Å) 45-1.7 Pseudo-Free CC (correct/ inverted) 80.03/54.25

Refinement PDB-ID 2VCL 2VCK 2VGR Resolution (Å) 38-1.55 (1.59-1.55) 49-1.80 (1.85-1.80) 48-2.1 (2.15-2.1) Rcryst(%) 17.4 (21.3) 18.4 (21.9) 22.65 (26.1) Rfree(%)c 19.4 (23.3) 22.5 (n/a) 28.5 (n/a) Number of atoms Protein 1731 6949 7010 Biliverdin IXα - 172 172 Water/Glycerol 225 (18) 575 339 Number of sidechains with alternate conformations 5 9 0

Average Bfactor Overall (Biliverdin IXα) 17.0 15.3 (21.9) 29.0 (44.2)

Ramachandran Plot d favoured 158 (88.3%) 651 (88.7%) 625 (83.9%) Additional allowed 21 (11.3%) 82 (11.2%) 111 (14.9%) Generously allowed 0 0 3 (0.4%) Disallowed 0 1 (0.1%) 6 (0.8%) Root mean square deviation from ideality (protein atoms) d

Bonds (Å) 0.018 0.014 0.012

Angles (degrees) 1.73 1.47 1.52

a Data in parentheses represent values in the highest resolution bin. b For definition of Rmeas and Rmrgd-F see (38). c Rfree calculated from 5% of data omitted either randomly (2VCL) or in thin shells (2VCK, 2VGR) from refinement. d Calculated with Procheck (39)

12

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

FIGURE LEGENDS Figure 1: The biosynthesis of phycoerythrobilin (PEB) and phycocyanobilin (PCB). PEB biosynthesis proceeds via two different pathways. PebA and PebB catalyze consecutive two-electron reductions of biliverdin IXα (BV) and 15, 16-dihydrobiliverdin (DHBV) to yield phycoerythrobilin (PEB). PebS catalyzes the four-electron reduction of BV to PEB via the two-electron reduced intermediate 15, 16-DHBV. PcyA catalyzes a four-electron reduction of biliverdin IXα (BV IXα) to phycocyanobilin via the intermediate 181, 182- DHBV. The electrons for all reactions come from reduced [2Fe-2S] ferredoxin (Fdred). The carbons of the respective reduction sites are numbered. P = propionate side chain. Figure 2: Structure of PebS with bound substrate BV (SePebS-BV, 2VCK, chain C). The protein is shown in cartoon representation colored from blue to red starting from the N-terminal end (N) (residue 21) to the C-terminus (C). BV is shown in stick representation. The secondary structure elements are consecutively numbered with H# for helices and S# for strands. A short disordered loop between strands S1 and S2 is indicated by blue dots. Figure 3: Stereoview of protein conformations observed in the crystal structures of PebS. Shown are Cα-traces of the superposed monomers of SePebS without substrate (grey), SePebS-BV with bound substrate (red, chains A-D) and wild type PebS-BV with bound substrate (blue, chains A-D). Residue numbers are added in regular intervals. Figure 4: Stereoview of PcyA superposed onto PebS. PcyA (2D1E, yellow cartoon) has been superposed onto PebS (SePebS-BV, 2VCK, chain C, red cartoon) in PyMOL (RMSD 4.4 Å for 135 Cα atoms).

Figure 5: Stereoview of the active site of PebS with bound BV and putative H-bonds from the distal side. Shown are chains C (panel A) and D (panel B) of SePebS-BV and chains B (panel C) and A (panel D) of PebS-BV. BV (grey) is oriented with the A-ring left and the D-ring right. BV and active site residues (salmon) in the 3.5 Å sphere of BV are represented as stick models with nitrogen atoms in blue and oxygen atoms red.

13

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 1. Dammeyer et al.

14

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 2. Dammeyer et al.

15

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 3. Dammeyer et al.

16

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 4. Dammeyer et al.

17

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 5. Dammeyer et al.

18

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from

Thorben Dammeyer, Eckhard Hofmann and Nicole Frankenberg-Dinkelbiliverdin-complex and the substrate free form

Phycoerythrobilin synthase (PebS) of a marine virus crystal structures of the

published online July 28, 2008J. Biol. Chem. 

  10.1074/jbc.M803765200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2008/08/25/M803765200.DC1

by guest on June 15, 2018http://w

ww

.jbc.org/D

ownloaded from