Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins♦

Background: Cryptophytes like Guillardia theta utilize soluble phycobiliproteins for light-harvesting. Results: Guillardia theta adopted phycoerythrobilin biosynthesis from cyanobacteria, and the phycobiliprotein lyase GtCPES provides structural requirements for transfer of this chromophore to a specific cysteine residue of the apophycobiliprotein. Conclusion: Phycobiliprotein synthesis in Guillardia theta combines proven and novel components. Significance: Results provide a better understanding of the evolution and function of unusual phycobiliproteins in cryptophytes. Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded β-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed.

Once synthesized, the phycobilin is bound by a specific PBP lyase, which then facilitates the ligation of the chromophore to a specific cysteine residue within the apo-PBP (22)(23)(24). PBP lyases are distinguishable in the clades of E/F-, S/U-, and T-type lyases, and some members of the E/F-type have an additional isomerase function (25)(26)(27).
Because of their evolution by secondary endosymbiosis, cryptophytes are extraordinary organisms. They are derived from a eukaryotic ancestor host cell that engulfed a red algal cell. The red alga was reduced to a complex plastid within the cryptophyte cell (28,29). Hence, cryptophytes possess the following four genomes: the endosymbiont-derived plastidial and nucleomorph genomes and the mitochondrial and host nuclear genome. Many of the endosymbiotic genes involved were transferred to the host nucleus during evolution (30). Cryptophytes retained the capability of performing oxygenic photosynthesis like red alga or cyanobacteria, but their light-harvesting machinery has been extensively modified as demonstrated by their unusual PBPs. Hence, biosynthesis and assembly of PBPs are largely unexplored in cryptophytes. With the release of the G. theta nuclear genome sequence in 2011, a wealth of information has been made available to study these processes (31). About 51% of the nucleus-encoded proteins are unique, and 49% have homologs in other organisms. For instance, the cryptophycean PBP ␣-subunits share no homology to those of cyanobacteria or any other known proteins (32). In this study, we investigated the similarities and differences of PBP synthesis and assembly in cryptophytes compared with cyanobacteria. With the help of BLAST analyses of the different genomes of G. theta, we found several genes encoding proteins potentially involved in phycobilin biosynthesis and PBP assembly. We give first insights into cryptophycean PEB biosynthesis, which seems to be adopted from cyanobacteria. Moreover, we present the first crystal structure of a eukaryotic PBP lyase, the CpeS lyase from G. theta (GtCPES). This lyase was also characterized in terms of phycobilin specificity, affinity, and binding kinetics. GtCPES belongs to the clade of S/U-type lyases and is restricted to binding of DHBV and PEB.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were American Chemical Society grade or better unless specified otherwise. All assay components were purchased from Sigma. PreScission protease and expression vector pGEX-6P-1 were obtained from GE Healthcare; pASK-IBA7ϩ was from IBA, and pGro7 was from TaKaRa. Protino glutathione-agarose 4B from Macherey-Nagel, Strep-Tactin-Sepharose from IBA, and TALON metal affinity resin from Clontech were used. HPLC-grade acetone, acetonitrile, formic acid, and spectroanalytical grade glycerol were obtained from Mallinckrodt Baker. Sep-Pak cartridges were obtained from Waters. BV was obtained from Frontier Scientific, Carnforth, Lancashire, UK.
Construction of Expression Vectors and Site-directed Mutagenesis-For construction of most plasmids, synthetic genes that had been codon-optimized for Escherichia coli K12 (GENEius algorithm, MWG Eurofins Operon) were employed. The synthetic genes were PCR-amplified from vectors supplied by MWG Eurofins Operon containing the appropriate sequence with primers encompassing selected recognition sites for cloning into an appropriate host vector. The plastid-encoded HO from G. theta was PCR-amplified from total genomic DNA, and the corresponding PCR product was also cloned into a host vector. Expression plasmids, the corresponding primers, recognition sites, the host vectors, and GenBank TM accession numbers of the used synthetic genes are summarized in Table 1. All site-directed variants of GtCPES were generated from pGtCPES using the QuikChange site-directed mutagenesis kit (Stratagene) with help of primers listed in Table 1 (shown is only the forward primer; the reverse primer is the complement, and introduced base pair changes are underlined). The resulting plasmids were verified by sequencing. The construction of the plasmid pho1pebS was described before (20). Production and Purification of Recombinant Proteins-A culture of E. coli BL21 (DE3) carrying the respective expression plasmid was incubated at 37°C and 150 rpm in LB medium (GtHo and GtPEBA, supplemented with 100 mM sorbitol and 2.5 mM betaine) to an A 578 nm of 0.6. For production of GtCPES co-expression of chaperones, GroEL and GroES (pGro7; TaKaRa) were induced by addition of 0.5 mg/ml L-arabinose, and cells were grown to an A 578 nm of 0.7-0.8. Subsequently, cells were induced with isopropyl ␤-D-thiogalactopyranoside (0.5 mM; pGEX-6P1 derivatives) or anhydrotetracycline (200 ng/ml; pASK-IBA7ϩ derivatives) and incubated overnight at 17°C (GtPEBA and GtCPES) or 30°C (GtHO and GtPEBB). Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol), and disrupted by two passages through a Constant Systems Cell Disruptor at 35,000 p.s.i. (GtHo, GtPEBA, and GtPEBB) or two times with a 2.5-min sonication (GtCPES). After separation of cell debris, the supernatant was loaded onto a column containing Protino glutathione-agarose 4B (Macherey & Nagel; GtPEBA and GtPEBB) or a Strep-Tactin-Sepharose (IBA; GtCPES). Purification was carried out according to the manufacturer's instructions based on sodium phosphate buffer (60 mM sodium phosphate, 300 mM NaCl, pH 7.5; GtCPES), potassium phosphate buffer (100 mM potassium phosphate, 100 mM NaCl, pH 7.2; GtHo) or TES/KCl buffer (25 mM TES, 100 mM KCl, pH 7.5; GtPEBA and GtPEBB). If required, purified GST-GtPEBB was incubated with PreScission protease (GE Healthcare) and dialyzed against cleavage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT). Tag-free GtPEBB was then obtained by a second affinity chromatography. Purified proteins were dialyzed three times against appropriate buffers mentioned above and concentrated using Vivaspin 6 concentrators (molecular mass cutoff 10,000 Da; Sartorius Stedim). Concentrations of proteins were determined using the calculated molar extinction coefficient ⑀ 280 (33).
Co-expression Experiments-Heterologous co-expression of GtCPES and PEB biosynthesis enzymes was performed in E. coli BL21(DE3) containing pET-GtCPES and pho1pebS. Cultures were grown in LB medium supplemented with 100 mM sorbitol and 2.5 mM betaine at 37°C, 150 rpm to an A 578 nm of 0.6 prior to induction with 0.5 mM isopropyl ␤-D-thiogalactopyranoside and incubated overnight at 17°C (16 h). Afterwards, cells were harvested, and the blue-colored cells were disrupted by sonication, and the supernatant was separated from cell debris by centrifugation. The His 6 -GtCPES⅐PEB complex was purified by affinity chromatography using TALON metal affinity resin (Clontech). The purification was carried out according to the manufacturer's instructions based on sodium phosphate buffer, pH 7.5.
Analysis of HO and FDBR Activity-After expression of GtHo in E. coli BL21 (DE3), the green-colored cells were disrupted using a Constant Systems Cell Disrupter at 35,000 p.s.i., and cell debris and supernatant were separated by centrifugation. The supernatant was diluted 1:10 in 0.1% TFA to precipitate proteins. Pigments were extracted using a Sep-Pak Light C18 cartridge, concentrated, and dried with help of a SpeedVac concentrator. The isolated phycobilins were analyzed via HPLC as described previously (21).
Anaerobic bilin reductase assays were performed utilizing an Agilent 8453 UV-visible spectrophotometer as described previously (35) with the following modifications. Reaction mixtures were incubated at 20°C for 10 min, and the reaction was followed spectroscopically every 30 s. Phycobilins were extracted using a Sep-Pak Light C18 cartridge, concentrated, and dried with the help of a SpeedVac concentrator. The isolated phycobilins were analyzed via HPLC.
GtCPES-Phycobilin Binding Studies-Equimolar amounts (10 M) of PBP lyase and phycobilin were mixed in sodium phosphate buffer, pH 7.5. Absorption spectra (Agilent 8453 UV-visible spectrophotometer) of the mixture and free phycobilins in the same buffer were compared.
Stopped Flow Kinetics-For stopped flow experiments, an SFM-400 apparatus with MOS-200 optics was used (Bio-Logic). Measurements were performed at 20°C in sodium phosphate buffer, pH 7.5. Different concentrations of GtCPES were rapidly mixed with a constant amount (2 M) of DHBV, 3(E)-PEB, or 3(Z)-PEB, and the increase in the absorption maximum was detected at 600 nm (PEB) or 610 nm (DHBV). Six to eight time traces were accumulated and averaged, and an exponential equation was fitted to the experimental data yielding the k obs value for each concentration. All experiments were repeated twice.
Isothermal Titration Calorimetry-Isothermal titration calorimetry (ITC) for analyzing GtCPES/PEB interaction was performed at 20°C in sodium phosphate buffer, pH 7.5, using an isothermal titration microcalorimeter (MicroCal Auto-iTC200, GE Healthcare). The temperature-controlled sample cell contained 40 M 3(E)-or 3(Z)-PEB, respectively, and GtCPES (400 M) was injected in 1.7-l steps, whereupon the change in heating power was recorded over 180 s until equilibrium was reached after each injection. Background heat generation from dilution of PEB or GtCPES in buffer was subtracted and fitted to each measurement. Measurements with 3(E)-PEB were averaged over two ITC experiments.
Size Exclusion Chromatography-The oligomerization state of proteins was determined using a GE Healthcare Superdex 200 HR10/300 GL size exclusion column. The column was equilibrated in appropriate buffer at a flow rate of 0.5 ml/min. Standards with known molecular weight (i.e. alcohol dehydrogenase, 150,000; bovine serum albumin, 66,000; carbonic anhydrase, 29,000; cytochrome c, 12,400) were applied to the column, and their elution volumes were determined spectroscopically at 280 nm. For chromatography of GtCPES sodium phosphate buffer, pH 7.5, of GtHo potassium phosphate buffer, pH 7.2, and of GtPEBA and GtPEBB, TES-KCl buffer at pH 7.5 was used.
Data Collection and Structure Determination-The x-ray diffraction data of native GtCPES and SeMet-GtCPES were collected at the Swiss Light Source (SLS) in Villigen, Switzerland, on beamline X10SA. All data were processed and scaled using XDS and XSCALE (40). Phenix.autosol (41) was used to obtain phases, searching for four selenium positions up to a resolution of 4.3 Å with phase extension to 1.95 Å using the native dataset. Phenix.autobuild calculated an initial model that was manually improved using Coot (42). The structure was refined using phenix.refine (41) against experimental phases and the native dataset. Refinement included individual B-factors and hydrogens. Data, model, and refinement statistics are given in Table 2. Although the data between 2.0 and 1.95 Å still improve model refinement, but fall below the classically used criteria for resolution limits, we use 2.0 Å as the resolution of the structure throughout this work.

RESULTS
Phycobilin Biosynthesis in the Cryptophyte G. theta-To obtain insights into phycobilin biosynthesis in the cryptophyte G. theta, the potentially involved plastid encoded heme oxygenase GtHo, 4

and the nucleus-encoded FDBRs
GtPEBA and GtPEBB were heterologously produced in E. coli and subsequently examined with regard to their enzymatic activity.
The plastid-encoded GtHo shares 51% identity with HO1 from Synechocystis sp. PCC6803, but only 12% identity with HY1 and HO3 from Arabidopsis thaliana. Heterologously produced recombinant GtHo was found to bind heme but was inactive in in vitro HO assays (data not shown). Gel permeation experiments indicated that inactivity of GtHo was likely due to aggregation of the protein, which was presumably caused by incorrect protein folding. Based on these results, intensive efforts to optimize the production of recombinant GtHO were made. Expression of StrepII-tagged GtHo in E. coli in LB medium supplemented with the osmolytes betaine and sorbitol led to green-colored cells, indicating that StrepII-GtHo was able to convert heme produced by E. coli yielding the green-colored open-chain tetrapyrrole BV IX␣ in vivo.
To confirm the identity of BV IX␣, the cells were disrupted, and open-chain tetrapyrroles were extracted and analyzed via HPLC (Fig. 1). The extracted pigment eluted with a retention time of 37.8 min, which resembles that of commercially available BV IX␣. Therefore, GtHo is an ␣-meso carbonspecific HO. The PBP PE545 uses DHBV associated with the ␣-subunits and PEB attached to the ␤-subunits as light-harvesting chromophores. We therefore presumed the presence of at least one gene encoding an FDBR. In fact, two genes encoding putative FDBRs were identified in the nuclear genome of the cryptophyte G. theta. Because of their eukaryotic origin, synthetic genes adapted to the codon usage of E. coli K12 (GENEius, MWG Eurofins Operon) were used for construction of expression plasmids. The corresponding proteins were designated GtPEBA and GtPEBB due to tBLASTn searches and sequence identities of 31.5% to PebA and 29% to PebB of Synechococcus sp. WH8020, respectively. Both proteins were heterologously produced in E. coli as GST fusion proteins. GST-GtPEBA was found to form inactive aggregates, and GST-GtPEBB was purified to homogeneity after removal of the GST tag. To probe their catalytic activities, these putative FDBRs were analyzed using an anaerobic bilin reductase assay as described for other FDBRs (36). GtPEBB did not bind or reduce BV IX␣, and only unspecific degradation of the bilin was observed ( Fig. 2A). In contrast, GtPEBB was able to bind DHBV as indicated by a shift of the absorption maxima of 335 and 561 nm of free DHBV to 340 and 606 nm after a short incubation time (Fig. 2B, UV-Vis). Immediately after starting the reaction, an increase of absorbance at ϳ670 nm was observed, which disappeared in the course of the reaction and is probably due to the formation of the substrate radical intermediate as shown for other FDBR enzymes (35,36,43). The absorbance of bound DHBV decreases during the reaction concomitant with the formation of a product with an absorption maximum at 535 nm. In subsequent HPLC analysis, the reaction product was identified as a mixture of 3(E)-and 3(Z)-PEB (Fig. 2B, HPLC), confirming the enzyme's PEB:ferredoxin oxidoreductase activity.
PebA from cyanobacteria is thought to transfer the intermediate DHBV to PebB without releasing it to the solvent via metabolic channeling (21). As no active PebA-like enzyme from G. theta was available, PebA from Synechococcus sp. WH8020 (SynPebA) was used to perform a coupled anaerobic bilin reductase assay employing GtPEBB (Fig. 2C). Addition of the NADPH-regenerating system to start the reaction resulted in a decrease of absorbance at ϳ690 nm followed by an increase of absorbance at ϳ590 nm due to conversion of BV IX␣ to DHBV by SynPebA. GtPEBB was added to the reaction mixture after complete production of DHBV and the formation of a substrate radical intermediate, and subsequently, PEB was observed (Fig.  2B). Hence, GtPEBB is able to functionally replace its cyanobacterial counterpart and act together with SynPebA in PEB formation.
These results give first insights into phycobilin biosynthesis in cryptophyte algae. The plastid-encoded GtHO provides the open-chain tetrapyrrole BV IX␣ as a substrate for further reduction steps by two FDBRs. Although an additional function of GtPEBA cannot be ruled out, it is most likely that GtPEBA converts BV to DHBV, the substrate of GtPEBB. GtPEBB reduces DHBV to PEB, the chromophore attached to the ␤-subunits of PE545.
PBP Lyase Homologs in G. theta-The covalent attachment of phycobilin chromophores to apo-PBPs in cyanobacteria is mediated by PBP lyases. Although spontaneous binding of phycobilins to the apo-PBPs can be observed, the lyases ensure the correct binding of the chromophore with regard to the specific attachment site and stereospecificity (24,44,45). As of 2014, only one eukaryotic lyase has been studied in detail. The open reading frame orf222 of chromosome 1 of the nucleomorph genome of G. theta was found to encode a T-type lyase (GtCPET), which is able to complement the function of its homolog slr1649 from Synechocystis sp. PCC 6803 by attaching PCB to Cys-␤155 of phycocyanin (46). Because G. theta does not use PCB in its PE545, the PBP lyase GtCPET seems to have a low substrate specificity. Expressed sequence tags (EST) encoding two additional putative PBP lyases were mentioned in the literature as follows: a phycocyanin ␣:PCB-like lyase (GtCPCX: GenBank TM accession number CAH25359.1 and KJ676834) and a CpeZ-like lyase (GtCPEZ: GenBank TM accession number CAJ73184.1 and KJ676835) (47,48). GtCPCX shows low homology to any characterized lyase but possesses a Huntington, elongation factor (EF3), protein phosphatase 2A (HEAT)-repeat domain typical for E/F-type lyases and a predicted Armadillo-type fold. The putative lyase GtCPEZ can be classified into the CpeZ family, which also belongs to the E/Ftype lyases (49). However, GtCPEZ shares only 18% identity with the only characterized member of this family (50). Furthermore, GtCPEZ lacks the HEAT-repeat domain, but an Armadillo-type fold can also be predicted. In 2011, the nuclear genome data of G. theta were released by the Department of Energy Joint Genome Institute (31). tBLASTn searches confirmed the localization of genes encoding GtCPCX and GtCPEZ in the nuclear genome. In addition, with GtCPES (GenBank TM accession numbers EKX47022.1 and KJ676836), we were able to identify a fourth putative PBP lyase, which was assigned to the clade of S/U-type lyases.
Purification and Co-expression of Recombinant PBP Lyases of G. theta-To investigate the attachment of light-harvesting chromophores to the PBPs of the cryptophyte alga G. theta, synthetic genes adapted to the codon usage of E. coli K12 (GENEius, MWG Eurofins Operon) encoding the putative lyases were used to construct appropriate (co-)expression vectors. GtCPCX, GtCPEZ, and GtCPET fusion proteins were found to be insoluble, but StrepII-GtCPES was purified almost to homogeneity (Fig. 3A). Moreover, the co-expression of His 6 -tagged GtCPES with PEB biosynthesis enzymes in E. coli yielded intensely blue-colored cells, indicating the formation of His 6 -GtCPES:PEB. Indeed, a blue protein complex was purified by affinity chromatography. However, the elution fraction contained two proteins, namely His 6 -GtCPES (22.1 kDa) and copurified His 6 -PebS, which could not be removed by size exclusion chromatography (28 kDa; PEB biosynthesis enzyme PebS from co-expression) (Fig. 3A). One may assume that the blue color of the protein fraction is due to binding of PEB to PebS, but the absorption spectrum of the blue fraction (Fig. 3B) shared no similarity with the spectrum of the PebS⅐PEB complex (51), but rather to the Prochlorococcus marinus MED4 CpeS⅐PEB complex (52). The bilin was separated from GtCPES via simple denaturing of the protein by dilution in 0.1% TFA, indicating a noncovalent binding of the bilin. HPLC analysis of the isolated bilins confirmed the bilin as PEB (Fig. 3C). This provides an improved method for preparing large amounts of PEB isomers. Compared with the conventional PEB isolation after Glazer and Hixson (53) and Terry (37), co-expression of GtCPES and PEB biosynthesis enzymes in E. coli and subsequent preparation by denaturing the GtCPES⅐PEB complex and PEB isomer separation via HPLC is much faster, easier, and cheaper. Moreover, this procedure overcomes the use of harmful substances like acetone, methanol, and mercury.

GtCPES Binds DHBV, 3(E)-PEB, and 3(Z)-PEB in Vitro-
Because of the presence of hemO, PEBA, and PEBB genes, G. theta shows all prerequisites for direct synthesis of the phycobilins DHBV and PEB. GtCPES binds PEB co-produced in E. coli. S/U-type lyases are known to exhibit a low apoprotein and bilin specificity but a high specificity for attachment of chromophores to positions homologous to Cys-84 (24, 45, 52, 54 -56). GtCPES shares equal values of sequence identity to characterized PCB and PEB transferring S-type lyases (data not shown). Hence, we tested the bilin stereoselectivity with a broad range of bilins. Absorption spectra of free bilins were compared with those after addition of equal amounts (10 M) of GtCPES. Bilins are found to reside in a cyclic, helical conformation in solution resulting in characteristic absorption properties. Upon binding to a protein, the conformation and thereby the absorption properties of the bilin change (57,58). For instance, a shift to longer wavelength in combination with a more distinct absorption maximum indicates a more stretched conformation of the bilin (59). Incubation of BV IX␣ and PCB with GtCPES caused no change of the absorption properties of these bilins (data not shown). In contrast, addition of GtCPES to free DHBV (559 nm), 3(E)-PEB (538 nm), and 3(Z)-PEB (536 nm) resulted in a higher and more distinct absorption maximum at a longer wavelength as follows: GtCPES⅐DHBV 611 nm; GtCPES⅐3(E)-PEB 603 nm; and GtCPES⅐3(Z)-PEB, 600 nm (Fig.   3, D-F). The GtCPES⅐bilin complexes were nonfluorescent and no zinc-induced fluorescence was detectable suggesting a noncovalent binding of the bilin (data not shown). Comparison of the absorption maxima of the GtCPES⅐PEB complex after coexpression (Fig. 3B) is in agreement with those of the in vitro reconstituted GtCPES⅐3(Z)-PEB complex (Fig. 3F). This result identified 3(Z)-PEB as the bilin bound by GtCPES under native conditions, which is in agreement with findings for CpeS from P. marinus MED4 (52).
Dimeric GtCPES Binds DHBV and PEB with Similar Binding Kinetics and High Affinity-S-type lyases are found to function as monomers, homodimers, or heterodimers (52,55,56,60). Size exclusion chromatography of both apo-GtCPES and GtCPES⅐bilin complexes revealed comparable elution profiles with deducible relative molecular masses of about ϳ42 kDa, and it coincides with the calculated molecular mass of GtCPES homodimers (ϳ43 kDa). Titration experiments of GtCPES with DHBV, 3(E)-PEB, and 3(Z)-PEB indicated that two bilin molecules were bound by dimeric GtCPES (data not shown). Binding kinetics and thermodynamics of the rapid GtCPES⅐bilin complex formations were further investigated by stopped flow and ITC experiments. A constant amount of bilin was mixed with stepwise increasing concentrations of GtCPES, and increase of absorption was monitored at 611 nm (DHBV) or 600 nm (PEB isomers). The observed rate constant k obs was obtained by fitting a single exponential function to the experimental data and plotted against the protein concentration (Fig. 4A). The association rate constant k on is represented by the slope of the linear regression. The results were averaged over two independent experiments, and the standard errors were in a range of 5% for the PEB isomers and 20% for DHBV. In case of the PEB isomers, similar k on values were observed (3(E)-PEB, 2.1 M Ϫ1 s Ϫ1 ; 3(Z)-PEB, 2.6 M Ϫ1 s Ϫ1 ; see Table 3), whereas association with DHBV (1.3 M Ϫ1 s Ϫ1 ) was slightly slower. However, the association rate constants were in a similar range, and the lower k on value of DHBV can only be a vague indication for a faster binding of PEB in vivo.
The thermodynamics of the GtCPES⅐bilin complex formation was analyzed by ITC. Because of insufficient DHBV amounts, the binding affinity of GtCPES to DHBV could not be determined. ITC measurements were performed in duplicate with 3(E)-PEB and once with 3(Z)-PEB. The averaged thermodynamic parameters are summarized in Table 3. A representative ITC measurement with 3(E)-PEB is shown in Fig. 4B. The GtCPES Crystal Structure-GtCPES is the first biochemically characterized eukaryotic PBP lyase. To obtain insights into substrate binding and reaction mechanism of this S-type lyase with high specificity toward DHBV and PEB, the x-ray crystal structure was determined at 2.0 Å resolution. Data, model, and refinement statistics are given in Table 2. The atomic structure of GtCPES displays a 10-stranded, antiparallel ␤-barrel with a central internal cavity (Fig. 5A). The bottom of the ␤-barrel is closed off by the N-terminal ␣-helix ␣ 1 , whereupon helix ␣ 0 is formed by the N-terminal StrepII-tag. A third ␣-helix is inserted between strands ␤ 2 and ␤ 3 near the open side of the ␤-barrel. Strands ␤ 5 and ␤ 6 are relatively short. Because of flexibility of some loop regions, the GtCPES atomic structure lacks residue Phe-25 and residues 104 -108. Generally, loop regions, particularly toward the opening of the ␤-barrel and around the missing amino acids, show considerably higher B-factors than the average B-value. GtCPES belongs to the structural family of FABP within the superfamily of calycins (15,63). Members of this widely distributed superfamily are known to bind small, hydrophobic molecules in the barrel interior (15,64). For instance, they are involved in diverse transport and signal transduction processes and serve as a protein matrix for pheromones or coloring substances (65,66).
GtCPES was found to form homodimers in gel permeation experiments as well as in the crystal (Fig. 5B). The crystal structure revealed a disulfide bond between the Cys-149 residues of two GtCPES monomers. This cysteine residue intercepts strand ␤ 9 by forming a ␤-bulge within the strand. To test whether this disulfide bond is essential for dimer formation, gel permeation experiments were repeated under reducing conditions and with a C149A protein variant. In both cases, the elution behavior was unchanged (data not shown), demonstrating that the disulfide bond is not essential for dimerization. Furthermore, the C149A variant fully retained its phycobilinbinding ability (Table 4). Interface analysis using PDB ePISA (67) ranked the dimer contact around the disulfide bond first with an interaction area of 1077 Å 2 . Two additional interfaces observed in the crystal between the openings of the funnels of two molecules (1012 Å 2 ) and between ␣-helices ␣ 0 and ␣ 1 of one and ␣ 2 of a second molecule (496 Å 2 ) are classified with a lower score. In addition, GtCPES superposes well with the other structurally known PBP lyase TeCpcS (PDB code 3BDR (68)) both on the monomer level (root mean square deviation of 1.32 Å for 106 superposed C ␣ atoms), but also on the level of the proposed biologically active dimers (root mean square deviation of 1.90 Å for 284 aligned C ␣ atoms).
Based on the classification as a member of the FABP family and the functional data available for TeCpcS, the actual binding site of PEB can be expected to lie within the funnel of GtCPES. The crystal structure showed additional density within this region, which was modeled as 1,6-hexanediol from the crystal-

TABLE 3 Kinetic and thermodynamic parameters of GtCPES⅐bilin complex formation
Association rate constant (k on ) was obtained by stopped flow experiments and stoichiometry value (N), association constant (K a ), enthalpy (⌬H 0 ), and entropy change (⌬S 0 ) via ITC measurements. From those values, K d ϭ 1/K a and k off ϭ k on ϫ K d were calculated. ITC results with 3(E)-PEB were averaged over two measurements; ITC with 3(Z)-PEB was performed once due to insufficient amounts of bilin. ND means not determined.  lization buffer. Interestingly, 1,6-hexanediol is coordinated by the side chains of amino acid residues Arg-146, Glu-136, and Arg-148, which are highly conserved among S-type lyases (Fig.  5, C and D). To verify their role in phycobilin binding, GtCPES variants E136A, R146A, and R148A were generated and analyzed. Indeed, absorption spectra of free DHBV, 3(E)-PEB, and 3(Z)-PEB did not change significantly after incubation with the GtCPES variants E136A and R146A (Table 4), indicating a loss of binding capability of phycobilins in these two variants. In contrast, variant R148A showed absorption shifts comparable with those of wild type complexes.

DISCUSSION
PEB Biosynthesis in G. theta Adopted from Cyanobacteria-In addition to chlorophyll a and c 2 -containing antenna complexes, the cryptophyte G. theta uses the PBP PE545 for lightharvesting. The ␣-subunits of PE545 are associated with the chromophore DHBV, whereas the ␤-subunits carry three molecules of PEB (14). In cyanobacteria, DHBV represents the intermediate of PEB biosynthesis, but it has not yet been identified as a protein-bound chromophore. Although the crystal structure and the identity of the bound chromophores of PE545 have been known since 1999, phycobilin biosynthesis and the assembly of PBP in cryptophytes are poorly understood. Here, we provide the first insights into PEB biosynthesis in the cryptophyte G. theta. We identified an enzymatically active, plastidencoded HO, specific for cleavage of the ␣-meso carbon bridge of heme. GtHo provides the substrate BV IX␣, which is further reduced to PEB by two nucleus-encoded FDBRs. The enzymatic activity of the first FDBR, GtPEBA, could not be shown experimentally, but the second FDBR, GtPEBB, was found to reduce DHBV to PEB exclusively. Like other FDBRs (35,36), GtPEBB seems to act via a substrate radical mechanism. Furthermore, GtPEBB is able to bind and convert DHBV produced by SynPebA in vitro indicating the capability to perform metabolic channeling (21). Although another or an additional function of GtPEBA cannot be ruled out, a DHBV:ferredoxin oxidoreductase function for conversion of BV IX␣ to DHBV is most likely, because DHBV is the sole substrate of GtPEBB, and no genes encoding other FDBRs were found in the completely sequenced genome of G. theta. Taken together, even though the cryptophyte G. theta utilizes a modified PBP distinct from cyanobacterial ancestors, it apparently has retained the PEB biosynthesis machinery from cyanobacteria.
PE545 Assembly in G. theta-The attachment of phycobilins to apo-PBPs is usually mediated by PBP lyases that in general have high attachment site specificity (24). Although cyanobacterial PBP lyases have been investigated for some time, those of cryptophytes are largely unexplored.
Overall, the PBP PE545 of G. theta has to be assembled with four bilin chromophores, each of which is covalently linked to one or two specific cysteine residues (Fig. 6). Although the ␤-subunit harbors three PEB molecules at position Cys-␤50/61, Cys-␤82, and Cys-␤158, the ␣-subunit carries only one chromophore (DHBV) at position Cys-␣19. Prior to this study, the only identified PBP lyase of G. theta is encoded by orf222 on the nucleomorph genome and is the T-type lyase GtCPET (46). This lyase has a low substrate specificity and is involved in the attachment of PEB to position Cys-␤158 (46). GtCPES investigated in this study is likely involved in the chromophorylation of Cys-␤82. Unfortunately, due to insoluble recombinant apo-GtCPEB protein, we were unable to show this transfer directly (data not shown).
Thus far, the lyase(s) involved in the attachment of the doubly linked PEB molecule at position Cys-␤50/61 is thus far unknown. The likely candidate would be GtCPCX and/or GtCPEZ. Furthermore, one of these lyases (or even both) might be involved in the chromophorylation of the ␣-subunit GtCPEA. Alternatively, this attachment could be autocatalytic (Fig. 6).
Eukaryotic PBP Lyase GtCPES-With GtCPES, we identified the first cryptophycean S/U-type lyase and provided not only biochemical but also structural insights into the function of this eukaryotic PBP lyase. Cyanobacterial S/U-type lyases occur in monomeric, homo-, or heterodimeric forms and show high positional specificity for ligation of phycobilins to homologs of the residue Cys-84 (50,52,55,56,60). At the same time, their apoprotein specificity is low, because they mediate the chromophore transfer to this position of PBP ␣or ␤-subunits, with the exception of ␣-phycocyanin, which requires CpcEF lyases. Substrate specificity for different phycobilins varies among S/U-type lyases (50,52,55,56,60). For instance, the CpcS lyase from T. elongatus (TeCpcS) binds PCB and PEB as well as P⌽B (68), whereas PmCpeS from P. marinus is specific for binding of DHBV and PEB (52).
GtCPES was found to be homodimeric and to exclusively bind DHBV, 3(E)-, and 3(Z)-PEB. Neither BV IX␣ nor PCB was bound by GtCPES. The K D values (0.6 M) for the complex formation with both PEB isomers were similar and suggest a tight binding of the substrate, which is in line with findings for other PBP lyases (23,45,52,54,62). Based on our results, a covalent binding of the phycobilins by GtCPES comparable with TeCpcS (68) is unlikely. The slightly lower association rate constants for the formation of the GtCPES⅐DHBV complex compared with those of the GtCPES⅐3(E)-/3(Z)-PEB complex formation indicate PEB and the co-expression experiments in particular 3(Z)-PEB as the naturally bound substrates of  (52). Notably, FDBRs are known to produce predominantly the 3(Z)-phycobilin isomers in vivo (35). Therefore, it is likely that in G. theta, GtPEBB provides the 3(Z)-PEB isomer for attachment to the CpeB apoprotein by GtCPES and other lyases as well.
GtCPES Exhibits a Lipocalin-like Fold-The x-ray structure of the S-type lyase GtCPES from the cryptophyte G. theta consists of a 10-stranded antiparallel ␤-sheet displaying a modified lipocalin fold. Based on this structure, GtCPES is assigned to be a member of the FABP family within the structural superfamily of calycins. Calycins generally bind small hydrophobic molecules and carry out diverse functions (15,64). For instance, they act as transport molecules or play a role in olfaction or coloration of insects by binding and enhancing features of appropriate ligands (69,70). One example is the bilin-binding protein of the butterfly Pieris brassicae that binds biliverdin IX␥ leading to the blue color of the insect (66). Additionally, there are three more bilin-binding members of the calycin superfamily, whose crystal structures were recently published. UnaG originates from the Japanese eel Anguilla japonica, and its green fluorescence is based on a bound bilirubin molecule (71). Furthermore, the structure of the PBP lyase TeCpcS was determined within the National Institutes of Health Protein Structure Initiative in 2007 and published by Kronfel et al. (68). TeCpcS is a homodimeric universal PBP lyase capable of binding a wide range of phycobilins like PCB, PEB, and P⌽B, transferring them to residue Cys-84 of diverse ␣ and ␤ apo-PBPs (68). Utilizing the similarity to UnaG, which has been crystallized with bound bilirubin, a docking model for an extended PCB molecule in TeCpcS was described, which buries the bilin's D-ring in the barrel and exposes the A-ring on the barrel mouth. GtCPES shows 36% sequence identity with TeCpcS (Fig. 7A) and shares a similar overall structure (Fig. 8A). Furthermore, an identical homodimerization was observed for both lyases, but in the case of GtCPES, this interaction is not only formed by a large hydrophobic interaction site but is also strengthened by a disulfide bond. The third solved crystal structure is that of the PCBspecific PBP lyase CpcT from the cyanobacterium Nostoc sp. PCC7120 (72). Thus far, this is the first crystal structure of a PBP lyase with a bound chromophore. The overall structure of CpcT is similar to that of CPES and is also retained upon chromophore binding. However, arginine residues located at the opening of the binding pocket undergo major rearrangements when PCB is bound. The crystal structure furthermore revealed that PCB adopts a ZZZsss geometry in an M-helical conformation (72). Our spectroscopic data on the CPES⅐PEB complex would, however, rather suggest a more extended chromophore conformation. We therefore will have to await the crystal structure of the CPES⅐PEB complex to get an insight into PEB binding of CPES.
Glu-136 and Arg-146 of GtCPES Are Involved in Bilin Binding-To get a glimpse of the location of the PEB-binding site in the barrel interior, we exchanged conserved residues close to the bound artificial ligand 1,6-hexanediole. Binding studies with these GtCPES variants and different phycobilins revealed a participation of Glu-136 and Arg-146 in phycobilin binding. The importance of Arg-146 or homologous amino acid residues for phycobilin binding has previously been confirmed for TeCpcS and NCpcS-III (where N is Nostoc) (23,68). Interestingly, the TeCpcS-R151G variant still bound enough PCB to confer transfer to the apoprotein CpcB in E. coli (68). For Glu-136, the carboxylic function might be involved in positioning of the substrate by interaction with the tetrapyrrole nitrogens, as found for bilin-binding in FDBRs (51,73,74).
In the case of NCpcS-III, several other residues have been shown to be involved in PBP lyase activity (23). Two conserved tryptophan residues are noteworthy. Replacement of the conserved Trp-69 (homolog to GtCPES Trp-69, see Fig. 7) by a methionine residue resulted not only in a reduced binding capacity of NCpcS-III but also in a stereochemically incorrect attachment of PCB to the apoprotein CpcB (23). As Trp-69 is facing the ligand binding pocket, this suggests a participation in PEB binding by forminginteractions with ring B or C of the tetrapyrrole. Presumably, thestacking may be responsible for the stabilization of the ligand conformation and therefore the position of ring A, resulting in a stereochemically correct attachment to the apoprotein. NCpcS-III Trp-75 (homologous to GtCPES Trp-75) has been found to be only indirectly involved in activity (23). Although it is disordered in the TeCpcS structure, in our GtCPES structure Trp-75 is placed at the upper side of the GtCPES barrel, pointing toward the barrel center (Fig. 7B). Here, it might play a role during interaction with PE545 and could provide a kind of greasy slide for transfer of the substrate during the reaction.
Previous studies focused on PBP lyases like TeCpcS and NCpcS-III, which bind and transfer a broad range of phycobilins (PCB, PEB, and P⌽B) (23,68). In contrast, GtCPES is only capable of binding phycobilins with a reduced C15ϭC16 double bond (DHBV and PEB). The resulting single bond between C15 and C16 allows rotation of the D-ring and therefore a better adaptation to a tight binding pocket. In contrast, ring D of PCB is restrained to the tetrapyrrole plane by the C15ϭC16 double bond. When comparing the two barrel pockets of GtCPES and TeCpcS, two main differences are striking (Fig. 8). The general barrel diameter at the mouth is much narrower for GtCPES (21.3 Å versus 26.1 Å in diameter, Fig. 8B).
Although the diameter at the bottom of the barrel is nearly identical on the basis of the main chain (Fig. 8B), the bulkier hydrophobic side chains of residues Phe-123, Leu-89, Ile-65, and Met-67 of GtCPES lead to a restriction at the proposed position of the D-ring in the region of the critical residues Glu-136 and Arg-146 (Fig. 8C). The equivalent amino acid residues of TeCpcS (Leu-128, Pro-92, Ala-69, and Val-71) have substantially smaller side chains (Fig. 8D). These data would support the requirement of a flexible D-ring of the tetrapyrrole for efficient binding to the smaller binding pocket. In contrast, the more open binding pocket of TeCpcS would support the binding of a wider range of bilins. Manual docking of PEB into the GtCPES structure with energy minimization was unsuccessful, because residues Arg-148 and Met-67, for instance, restrict access to residue Arg-146, whose importance for PEB binding was demonstrated experimentally. Therefore, an induced fit of GtCPES upon ligand binding is likely.
GtCPES Structure Provides Requirements for Bilin Transfer to Cys-␤82-The bathochromic shift of the absorption spectrum and the formation of a more distinct absorption peak upon binding of PEB to GtCPES suggest a more or less stretched conformation (57,58) of the bound phycobilin in accordance with a binding mode similar to the model of PCB in TeCpcS. Phycobilins are usually linked to the apoprotein via a thioether bond between the ethylidene group of ring A of the phycobilin and a conserved cysteine residue of the apoprotein (24). Therefore, it is likely that ring A of PEB is exposed at the upper side of the ligand-binding site of GtCPES. The surface electrostatics of GtCPES and the ␤-subunit of PE545 from Rhodomonas sp. CS24 (which shares 94% sequence identity with the respective G. theta homolog) (14) suggested a potential docking site for the upper side of the GtCPES molecule to an area around Cys-82 of the PE545 ␤-subunit (Fig. 9). Indeed, a docking model consistent with this proposal was ranked first by the GRAMM-X Protein-Protein Docking Web Server (75). Hence, the area around PE545-Cys-␤82, particularly made up by positively charged and neutral amino acids (Fig. 9C), is likely to bind the negatively charged part of the upper site of GtCPES (Fig.  8D). In contrast, all conserved cysteine residues of the ␤-subunit are especially surrounded by negatively charged and neutral amino acids, and thus they would not provide an ideal binding surface for the negatively charged patches of GtCPES. This is indicative of a strong selection of Cys-82 by the S-type lyase GtCPES. However, we expect a considerable structural change in PE545, as Cys-82 is not directly accessible and needs to be exposed to the attachment of the chromophore by GtCPES. In the case of cyanobacterial PBP chromophorylation and assembly, it is postulated that the PBP subunits are folded prior to chromophorylation and subsequent assembly of the PBP oligomers (76). Therefore, it is likely that the tertiary structure of the PBP subunit is important for recognition by and docking of PBP lyases. . Proposed amino acid residues involved in phycobilin binding and PBP lyase activity in different S-type lyases. A, sequence alignment of different S-type lyases. Gt, G. theta; Pm, P. marinus MED4; Fd, Fremyella diplosiphon; N, Nostoc sp. PCC7120; Te, T. elongatus (PDB code 3BDR). Identical residues and those with 60% similarity are shown with colored background. Consensus: identical (*); conserved substitution (:); semi-conserved substitution (.). Residues involved in phycobilin binding in GtCPES, black arrows; residues involved in phycobilin binding and PBP lyase activity in NCpcS-III (red arrows); and TeCpcS (gray arrows). Corresponding homolog residues in GtCPES are blue, NCpcS-III red, and TeCpcS gray. B, GtCPES structure with residues essential for phycobilin binding (black) and homologs (blue) to important residues in NCpcS-III (red) and TeCpcS (gray).