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J. Biol. Chem., Vol. 281, Issue 37, 27081-27089, September 15, 2006

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Insights into Phycoerythrobilin Biosynthesis Point toward Metabolic Channeling^*

From the Institute for Microbiology, Technical University Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany

Received for publication, May 30, 2006 , and in revised form, July 19, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Together with other bilins such as phycocyanobilin it serves as a light-harvesting pigment in the photosynthetic light-harvesting structures of cyanobacteria called phycobilisomes. The biosynthesis of both pigments starts with the cleavage of heme by heme oxygenases to yield biliverdin IX, which is further reduced at specific positions by ferredoxin-dependent bilin reductases (FDBRs), a new family of radical enzymes. The biosynthesis of phycoerythrobilin requires two subsequent two-electron reductions, each step being catalyzed by one FDBR. This is in contrast to the biosynthesis of phycocyanobilin, where the FDBR phycocyanobilin: ferredoxin oxidoreductase (PcyA) catalyzes a four-electron reduction. The first reaction in phycoerythrobilin biosynthesis is the reduction of the 15,16-double bond of biliverdin IX by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3¹,3²-diene structure of 15,16-dihydrobiliverdin to yield phycoerythrobilin. Both FDBRs from the limnic filamentous cyanobacterium Fremyella diplosiphon and the marine cyanobacterium Synechococcus sp. WH8020 were recombinantly produced in Escherichia coli and purified, and their enzymatic activities were determined. By using various natural bilins, the substrate specificity of each FDBR was established, revealing conformational preconditions for their unique specificity. Preparation of the semi-reduced intermediate, 15,16-dihydrobiliverdin, enabled us to perform steady state binding experiments indicating distinct spectroscopic and fluorescent properties of enzyme·bilin complexes. A combination of substrate/product binding analyses and gel permeation chromatography revealed evidence for metabolic channeling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phycobilins are linear tetrapyrrole molecules that are important cofactors of the photoreceptor phytochrome and the cyanobacterial light-harvesting phycobiliproteins. One of the major pigments found in the phycobilisomes of certain cyanobacteria, red algae, and cryptomonads is phycoerythrobilin (PEB).³ In these organisms PEB is covalently linked to the phycobiliprotein phycoerythrin (PE), a major constituent of the light-harvesting structures called phycobilisomes. These structures allow the organisms to efficiently absorb light in regions of the visible spectrum that are poorly covered by chlorophylls. Through resonance energy transfer the absorbed light energy is transferred to the photosynthetic reaction centers in the membrane. Freshwater cyanobacteria of the genus Calothrix (Fremyella) are able to adapt their phycobiliprotein composition within the phycobilisome in response to different light conditions. In a process called complementary chromatic adaptation the organisms are able to adjust the quantities of phycocyanin and PE for maximal light harvesting efficiency. Not only is the synthesis of apophycobiliproteins and linker proteins regulated by light, but also the biosynthesis of the enzymes required for PEB synthesis. It has been demonstrated that the expression of the genes pebA and pebB encoding ferredoxin-dependent bilin reductases (FDBRs) in Fremyella diplosiphon (Calothrix or Tolypothrix sp. PCC 7601) is up-regulated by green light, as is the expression of the cpeBA genes encoding the - and -subunits of PE (1).

In a similar manner, marine cyanobacteria of the Synechococcus group are able to regulate phycourobilin to PEB ratios by adjusting the expression of phycoerythrins with different phycourobilin content, PE(I) and PE(II) (2), or, as recently suggested, by lyases that mediate PEB isomerization on the phycobiliproteins (3). Because these organisms retain a fixed phycocyanin:PE ratio (4), they are not considered as classical chromatic adapters.

The biosynthesis of phycobilins proceeds via the heme biosynthetic pathway. The final product, heme, is cleaved by heme oxygenases to yield biliverdin IX (BV), which is subsequently further reduced by a family of FDBRs (Fig. 1). These enzymes are characterized by a distinct double bond regiospecificity resulting in bilins with a wide variety of spectroscopic properties. Synthesis of phytochromobilin (PB), the chromophore of plant phytochromes, is catalyzed by phytochromobilin synthase (HY2) through a formal two-electron reduction at the A-ring 2,3,3¹,3²-diene structure. Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the four-electron reduction of BV to phycocyanobilin (PCB), the chromophore of certain cyanobacterial phytochromes and one of the major light-harvesting pigments in cyanobacterial phycobilisomes. PcyA is the best described member of the FDBR family. It mediates two subsequent electron reductions at both vinyl groups of BV (5). In this reaction 18¹,18²-dihydrobiliverdin is a visible semi-reduced intermediate. Because no metal or organic cofactors could be detected in the FDBR family, a radical mechanism for PcyA was postulated (5). Evidence for the appearance of an intermediate substrate radical was recently demonstrated by absorbance and EPR spectroscopy (6). Structural information to this new family of enzymes has recently been added through the solved crystal structure of the Synechocystis sp. PCC 6803 PcyA (7).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Biosynthetic pathway of bilin pigment synthesis. In plants, red algae, cyanobacteria, and cryptomonads BV is reduced by FDBRs with high double bond regiospecificity to produce phytobilins. PEB synthesis is catalyzed by PebA and PebB and proceeds via the semi-reduced intermediate DHBV.

Here we present the biochemical characterization of recombinant PebA and PebB from the filamentous freshwater cyanobacterium, F. diplosiphon, and the unicellular marine cyanobacterium Synechococcus sp. WH8020. From our results, the involvement of PebA and PebB in metabolic channeling is postulated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents—Unless otherwise specified, all chemical reagents were ACS grade or better. Spinacia oleracea ferredoxin, Clostridium pasteurianum ferredoxin, Porphyra umbilicales ferredoxin, ferredoxin:NADP⁺ oxidoreductase, glucose-6-phosphate dehydrogenase, and NADP⁺ were purchased from Sigma-Aldrich. Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs and Phusion^TM DNA polymerase from Finnzymes. HPLC grade acetone, chloroform, and formic acid were purchased from Acros, Sigma-Aldrich, and J. T. Baker, respectively.

Glutathione-Sepharose^TM 4FF, PreScission^TM protease, and expression vector pGEX-6P-3 were obtained from GE Healthcare. Expression vector pASK-IBA45⁺ and Strep-Tactin-Sepharose® were purchased from IBA. Stirred ultrafiltration cell and filters were obtained from Millipore.

Construction of pebA and pebB Expression Vectors—The sequences for pebA and pebB (GenBank^TM accession number AY363679 [GenBank] ) were amplified from chromosomal DNA of F. diplosiphon (Fredi) strain Fd33 (9) obtained from the laboratory of D. Kehoe. PCRs were set up using Phusion^TM DNA polymerase. The forward and reverse primers were: 5'-GGAATTCGATCTATAAGTGCTTCCTTGAGC-3' and 5'-CCGCTCGAGCTATTTGGCTACAACAGTTGCTAATG-3' for pebA; and 5'-GGAATTCGATCCGGAGCGAAGCGAAGTTG-3' and 5'-AACTGCAGTTATTTGATAGCTGATGTGAGCTTTC-3' for pebB (the underlined bases indicate the EcoRI, XhoI, or PstI sites). The PCR product pebAFredi was ligated into pGEX-6P-3 vector for N-terminal fusion with glutathione S-transferase (GST). The plasmid was transformed in Escherichia coli BL21(DE3) cells. The pebBFredi construct was ligated into pASK-IBA45⁺ for N-terminal fusion with Strep-tag® II and transformed in E. coli DH10B cells. The integrity of the plasmid constructs was confirmed by DNA sequencing. Cloning strategies for the Synechococcus sp. WH8020 (Synpy) pebA und pebB were described previously (10).

Production and Purification of Recombinant PebA and PebB—For production of recombinant PebAFredi, PebASynpy, and PebBSynpy, 2 liters of LB medium containing 100 µg/ml ampicillin was inoculated at 1:100 from an overnight culture of BL21(DE3) carrying the respective plasmid construct and cultivated at 37 °C to an A_{578 nm} 0.6–0.8. After a temperature shift to 17 °C, protein expression was induced by adding 100 µM isopropyl--D-thiogalactopyranoside, and cells were cultured for a further 15 h at 180 rpm. Cells were harvested by centrifugation and stored at –20 °C. Frozen cells were thawed, resuspended in 20 ml of lysis buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 0,05% Triton X-100), and disrupted by passage through a French press cell at 20,000 p.s.i. After ultracentrifugation for 50 min at 170,000 x g the supernatant was loaded on a glutathione-Sepharose^TM 4FF column. Washing and elution were done according to instructions supplied by the manufacturer. Protein-containing fractions were cleaved with 2 units of PreScission^TM protease/mg of protein in the recommended cleavage buffer. Cleavage led to an additional eight amino acid residues on the N termini of the proteins, and the first amino acid was changed from Met to Ile. A second glutathione-Sepharose column was used to separate the GST tag from the protein. The protein solutions were dialyzed against reaction buffer (25 mM TES-KOH buffer, pH 7.5, 100 mM KCl). If indicated, an additional purification step was performed using gel permeation chromatography on a High Load^TM 26/60 Superdex^TM 75 prep grade column (GE Healthcare). The proteins were concentrated using a stirred ultrafiltration cell with a molecular weight cut-off of 10,000 and stored up to 3 days on ice.

Production of recombinant PebBFredi was induced using anhydrotetracycline (200 µg/ml). Cultivation conditions (i.e. medium, temperature) were identical to those described above. Purification was done on a Strep-Tactin-Sepharose® column as recommended by the manufacturer. Strep-tagged PebBFredi is N-terminally extended by 20 additional amino acids (Strep-tag® II).

Purification of Recombinant Reductants—The DNA sequence of Synechococcus sp. PCC 7002 ferredoxin (petF) was amplified from the plasmid pSe280fd (obtained from D. Bryant) using the following forward and reverse primers: 5'-GGAATTCGATCGCTACATATAAGGTTAC-3' and 5'-CCGCTCGAGCTAGTAGAGTTCTTCCTCTTT-3' (the underlined sequences indicate the EcoRI or XhoI sites). The PCR product was ligated to pGEX-6P-3. Expression was done in NZCYM medium as described elsewhere (11), and protein production was induced using 1 mM isopropyl--D-thiogalactopyranoside at an A_{578 nm} of 0.6–0.8. Cells were harvested 4 h after induction. Purification was done with two sequential glutathione-Sepharose columns as described for the bilin reductases. The employed cleavage buffer was free of dithiothreitol and EDTA.

Determination of Protein and Bilin Concentrations—Concentrations of the bilin reductases were determined using the calculated molar extinction coefficient (_{280 nm}) (12).

The concentration of recombinantly produced ferredoxin from Synechococcus sp. PCC 7002 was determined using an _{420 nm} of 9.7 mM^–1 cm^–1 (13). Concentration of BV IX was calculated using an _{376 nm} of 68.6 mM^–1 cm^–1 and an _{698 nm} of 32.6 mM^–1 cm^–1 in 2.5% HCl-MeOH (14). The concentration of 3E-PEB was determined using _{326 nm} 15.8 mM^–1 cm^–1 and _{591 nm} 25.2 mM^–1 cm^–1 in 5% HCL-MeOH (15). The concentration of DHBV was determined by measuring the absorbance at the 561-nm maximum in 5% HCl-MeOH and using the long wavelength extinction coefficient of 3E-PEB for calculation. All concentrations were determined using a Ultrospec 2000 UV-visible spectrophotometer (GE Healthcare).

Bilin Reductase Activity Assay—Assays for bilin reductase activity were done as described previously with small modifications (10). The standard assays contained 1.5 µM bilin reductase, 5 µM bilin substrate, and 4.8 µM recombinantly produced Synechococcus sp. PCC 7002 ferredoxin or the alternative ferredoxins in reaction buffer. The assays were incubated for 15–30 min at 30 °C in the dark. Bilins were isolated using C18 Sep-Pak columns (Waters) and evaporated to dryness in vacuo. For spectrometric detection of electron transfer activity, the assay was performed using 10 µM PebAFredi, 10 µM BV, 40 µM NADP⁺, and 0.0125 units/ml ferredoxin: NADP⁺ oxidoreductase.

Preparative Production of 15,16-Dihydrobiliverdin—Larger quantities of DHBV were produced enzymatically by setting up a 10-ml bilin reductase activity assay containing 20 µM BV, 5 µM PebAFredi, and 4.8 µM Synechococcus sp. PCC 7002 ferredoxin in reaction buffer at 30 °C. The reaction progress was monitored by measuring absorbance spectra at different time points during the reaction. If no further substrate conversion was observed, the reaction was stopped immediately by adding 40 ml of 0.1% (v/v) trifluoroacetic acid and cooling on ice. A C18 Sep-Pak light column was preconditioned with sequential washes of CH₃CN, H₂O, 0.1% (v/v) trifluoroacetic acid, and 10% (v/v) MeOH in 0.1% trifluoroacetic acid. The bilin was loaded on the column washed with 6 ml 0.1% (v/v) trifluoroacetic acid and 6 ml of 20% MeOH in trifluoroacetic acid, eluted with CH₃CN, and dried in vacuo. The purity of produced DHBV was controlled by HPLC for contamination by other bilins.

Absorption and Fluorescence Spectroscopic Analysis—All protein solutions used for binding site saturation experiments were checked for homogeneity with analytical gel permeation chromatography on a Superdex 75 HR10/30 column. Protein solutions were adjusted to concentrations ranging from 0.5 to 18 µM; substrate and product complexes were formed by incubating the protein solution with a 4 µM final concentration of the bilin (5–10 µl of stock solution) for 20 min on ice in the dark. All fluorescence measurements were performed under physiological conditions in reaction buffer using an Aminco Bowman AB2 spectrofluorimeter at a constant temperature of 20 °C. The excitation/emission wavelengths used were 590 nm/610 nm for PebA·DHBV, 605 nm/645 nm for PebB·DHBV, and 545 nm/630 nm for PebB·3E-PEB. Both excitation and emission slit widths were set at 4 nm, and the scan speed was 2.5 nm/s. Determination of binding constants of substrate/product to the enzymes was done according to Clarke (16). Binding curves were measured by the increase of fluorescence intensities, as the bilin·enzyme complex is formed at equilibrium. Obtained data were analyzed using Sigma Plot 9.0 (Systat Software Inc.), and data were fitted against Equation 1.

(Eq. 1)

Time-dependent absorbance measurements (Fig. 4) were performed in a stirred cell tempered to 30 °C on an Agilent Technologies 8453 spectrophotometer with ChemStation biochemical analysis software. Absorbance spectra (Fig. 5) were measured using a Lambda 2 UV-visible spectrophotometer (PerkinElmer Life Sciences).

HPLC-Analysis—Bilin reaction products were analyzed as described previously (5).

Gel Permeation Chromatography of Enzyme·Bilin Complexes—Enzyme·bilin complexes were formed by incubating protein solution with approximately double the molar concentration of bilin for 3 min at 30 °C. The complex was purified by passing it through a NAP^TM-5 column (GE Healthcare) prior to analytical gel permeation chromatography on a Superdex 75 HR10/30 column. During isocratic elution, absorbance was simultaneously detected at 280, 585, and 605 nm using the UV-900 detector of the ÄKTApurifier system (GE Healthcare).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Recombinant Production and Purification of FDBRs—PebA of the filamentous freshwater cyanobacterium F. diplosiphon and also PebA and PebB of the coccoid marine cyanobacterium Synechococcus sp. WH8020 were expressed using a tac promoter-driven N-terminal GST fusion protein. A protocol using overnight proteolytic cleavage of the affinity-purified GST fusion protein followed by a second affinity chromatography to remove GST tag and protease led to the best results. This purification strategy led to less than 10% impurity (Fig. 2). Prior to all quantitative experiments, a third purification step using gel permeation chromatography was performed to remove possibly aggregated enzyme. N-terminal sequencing of PebAFredi by Edman degradation revealed no GST or other protein contamination after this purification step, and the yields of this method varied, depending on the enzyme, between 3 and 7 mg/liter cell culture. The best results for PebBFredi purification were achieved with tet promoter-driven expression followed by single-step purification of the Strep-tagged enzyme (Fig. 2). This procedure yielded about 1 mg/liter cell culture.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Coomassie-stained SDS-PAGE of purified FDBRs. Three-step purified PebAFredi (1) and PebASynpy (3) after affinity and gel filtration chromatography. One-step affinity-purified Strep-tagged PebBFredi (2) and PebBSynpy after two affinity chromatography columns (4). M is the protein molecular mass marker.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. HPLC elution profiles of PebA and PebB metabolites. HPLC elution profiles of BV (top; 650 nm), and the reaction products of the PebA-catalyzed reaction, DHBV (middle; 560 nm) and PebB 3E- and 3Z-PEB (bottom; 560 nm). Enzyme assays and bilin extractions were performed as described under "Experimental Procedures." PEB products were produced using PebA and PebB simultaneously. Minor unassigned peaks are impurities in commercial BV (BV) and an unidentified non-enzymatic degradation product (DHBV).

View larger version (48K): [in this window] [in a new window]   FIGURE 4. Time-based spectroscopic monitoring of the PebA-catalyzed conversion of BV to DHBV. An enzyme assay containing 10 µM PebASynpy and the substrate BV (10 µM) was set up, and the reaction was started by the addition of an excess of reduced ferredoxin. Following the spectral changes during the reaction by measuring the spectra every 30 s displays the in vitro enzymatic conversion of BV to DHBV.

Substrate Specificity of FDBRs—To analyze the substrate specificities of the various FDBRs we examined different natural bilins (Table 1). In our standard HPLC assay system, we did not find 3E-PCB to be converted by PebA, indicating that a lack of A- and D-ring vinyl moieties, together with a changed geometry of the A-ring ethylidene group, prevents recognition of 3E-PCB as substrate. DHBV was not converted by PcyA, demonstrating that the reduction of the 15,16-double bond causes structural difference in the bilin, which likely prevents proper placement of the bilin in the active site pocket. Interestingly, we found that PebA was able to reduce the plant bilin PB to PEB, indicating that an A-ring ethylidene group instead of an A-ring vinyl group is not critical for substrate recognition by PebA.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Spectroscopic properties of bilins and enzyme·bilin complexes. Enzyme·bilin complexes were formed by incubating 8 µM FDBR with 4 µM bilin for 20 min on ice. A, absorbance spectra of BV (solid line), PebA·BV (dashed line), and PebB·BV (dashed-dotted line). B, absorbance spectra of DHBV (solid line), PebA·DHBV (dashed line), and PebB·DHBV (dashed-dotted line). C, absorbance spectra of 3E-PEB (solid line), PebA·3E-PEB (dashed line), and PebB·3E-PEB (dashed-dotted line). Formation of enzyme·bilin complexes caused intensive changes in absorbance properties of the bilins. The shoulder at 430 nm in the DHBV spectra (B) is possibly an indication of non-enzymatic rubin-like degradation products.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Fluorescence emission spectra of enzyme·substrate/product complexes. Fluorescence emission of enzyme·bilin complexes excited at their long wavelength absorbance maxima. Enzyme·bilin complexes were formed by incubating 8 µM FDBR with 4 µM bilin for 20 min on ice. Complexes of bilins with reduced 15,16-double bonds, like DHBV with PebA (solid line) or PebB (dashed line), as well as PebB with 3E-PEB (dash-dotted line), exhibit intensive fluorescence in the same intensity range. In contrast, the free bilins or the PebA·BV complex show only very weak or no fluorescence (data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Dependence of fluorescence emission intensity on protein concentration. Equilibrium binding experiments were performed as described under "Experimental Procedures." The addition of enzyme (in this example PebB) in concentrations that varied between 0 and 18 µM to 4 µM bilin (in this example DHBV) led to increased fluorescence emission. Fluorescence intensity displays a hyperbolic dependence on enzyme concentration (inset).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
PebA and PebB Belong to the FDBR Family of Radical Enzymes—In this current work we have presented the first detailed biochemical characterization of the two enzymes involved in PEB biosynthesis. They belong to the newly defined family of FDBRs. The best described member of this family is PcyA, which catalyzes the four-electron reduction of BV IX to PCB. Because of the lack of bound metal cofactors and the detection of a bilin radical intermediate, the family of FDBR constitutes a novel family of radical enzymes (6). Although we have not presented any EPR data, PebA lacks metal ion cofactors, and the reaction most likely proceeds via radical intermediates. Interestingly, we observed that DHBV bound to PebA can be reoxidized to BV by molecular oxygen. Reactive oxygen species such as peroxyradicals are known to reoxidize albumin bound bilirubin (BR) to BV (19). Although it seems unlikely that reactive oxygen species are produced under our experimental conditions, reactive oxygen species or molecular oxygen itself could serve as the oxidant. These observations fit the results in which a higher yield of a recombinant, in vivo produced PEB adduct of phytochrome (Cph1) was achieved under low aeration as reported recently (20). In this regard, PcyA has been described to reduce BV much more efficiently under anaerobic conditions (6). However, in our assay system removal of oxygen did not significantly enhance the rate of BV reduction, but the intermediate DHBV is more stable under anaerobic conditions (data not shown).

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Gel permeation chromatography elution profiles of purified PebA and PebB co-eluting with DHBV. A, purified PebA was incubated with its product, DHBV, and the complex was purified using a NAPTM5 desalting column prior to analytical gel permeation chromatography on a Superdex 75 HR10/30 column. The protein was detected by absorbance at 280 nm (solid thick line), and DHBV was detected at 585 nm (dotted line), corresponding to the absorbance maxima of PebA·DHBV, or 605 nm (solid thin line), corresponding to the absorbance maxima for PebB·DHBV. Binding was tight enough for coelution of enzyme and bilin (monomer). B, elution profile of the subsequent enzyme PebB (as a GST fusion dimer) without bound bilin. C, elution profile of purified PebA·DHBV complex incubated with an equimolar amount of GST-PebB shows that approximately half of the bilin was detected as coeluting with PebB after incubation.

PebA and PebB Form Distinct Complexes—Binding of substrate or product to the enzymes leads in all cases to a red shift of the _max2. Red shifts like that were reported previously to occur upon protonation of the basic pyrrolenic nitrogen atom within the tetrapyrrole ring structure (22, 23) or as a result of an enhanced conjugated -electron system of the bilin (23, 24). Because protonation is presumably the first step in the catalytic bilin reduction by PcyA (6), it is quite possible that during enzyme binding the positioning of the bilin close to a proton donating amino acid or the coordination by an electropositive ligand causes these red shifts. Interestingly, DHBV in acidic methanol displays a rather blue-shifted spectrum (data not shown). Therefore, the observed spectral effects cannot easily be assigned as due to protonation or geometrical changes. The massive changes in the _max2/_max1 ratio that occur during binding of DHBV to PebA or PebB suggest a more linearly stretched conformation of the DHBV bound to the enzyme compared with free DHBV (22). This fact is interesting in regard to the conformation of BV in the PcyA crystal structure, which is cyclic (7). The spectroscopic differences between PebA·DHBV and PebB·DHBV indicate that the protein environment, possibly the conformation or orientation of the tetrapyrrole, is different in both complexes.

Fluorescent Properties of PebA·Bilin and PebB·Bilin Complexes—Free bilins exhibit very low fluorescence, but it has long been known that the covalent adducts of bilins to proteins lead to fluorescent proteins with high fluorescence quantum yields (25, 26). Because of their fluorescent properties, phycobiliproteins, especially PEs, are frequently used for fluorescence labeling in biotechnological applications as well as for quantification of phytoplankton composition (27, 28). Phytochromes become intensely fluorescent when the bilin chromophore is hindered from undergoing photoisomerization either by steric hindrance caused by the protein environment (29) or because the chromophore lacks the 15,16-double bond, as in the case of plant apophytochrome reconstituted with PEB (25) or bacterial apophytochrome reconstituted with DHBV (30). DHBV bound to PebA or PebB shows distinct fluorescent properties. The occurrence of this fluorescence indicates that the bilins are held in a sterically tightly fixed conformation as is the case in PE. Furthermore, bridging and restriction of substituent mobilities could account for the high fluorescence (23). This observation implies that the binding mechanism to both enzymes is different as is the position of the 15,16-double bond and, accordingly, the A-ring vinyl moiety of the substrate to be reduced.

PebA and PebB May Be Involved in Metabolic Channeling—Most parts of the tetrapyrrole metabolism involve rather labile intermediates, which in aerobic solution would last only for short periods of time. A tight control is further necessary, as many tetrapyrroles are highly toxic. Metabolic channeling has been demonstrated as a way to avoid unwanted or harmful side-reactions, e.g. for biosynthesis of the early precursor, 5-amino-levulinic acid (31, 32). Here we present some indications that this also might be relevant for the stages of bilin biosynthesis.

The involvement of two enzymes in the biosynthesis of PEB was already inferred from the genomic localization of the encoding genes in an operon when the bilin reductases were first identified in 2001 (10). In F. diplosiphon this operon structure was confirmed and, it was shown that pebA and pebB are co-transcribed from the same promoter generating a polycistronic mRNA (1). The fact that two enzymes are necessary to yield one product (PEB) and the operon structure led to the postulation of potential metabolic channeling (10). Metabolic channeling would ensure direct transfer of the intermediate DHBV from PebA to PebB without releasing it into the bulk solvent. In this regard, it has been demonstrated previously that the conversion of DHBV to PEB is not the rate-limiting step in PEB biosynthesis (1). Furthermore, it has already been postulated that PB synthase might be involved in metabolic channeling with a heme oxygenase in plants (17). Our current results provide more evidence that the intermediate DHBV is not released from PebA but rather is transferred directly to PebB. The results of the transfer of DHBV from PebA to PebB, together with the tight binding of the product DHBV to PebA, led us to the suggestion that under the expected low substrate concentration in vivo, PebB may play an important role in releasing and taking over the intermediate from PebA. The lower binding affinity of PEB to PebB may demonstrate the release of the final product PEB from the enzyme; but it may also be result of differences in the ethylidene side chain geometry of the 3Z-compared with the 3E-isomer that we used for titration experiments.

Conclusion and Outlook—Our results presented here have provided new insights into phycoerythrobilin biosynthesis with hints that it may involve metabolic channeling. Current efforts in our laboratory are focused on combining co-immunoprecipitation with surface plasmon resonance spectroscopy and other biophysical methods to ultimately prove the existence of such complexes.

	FOOTNOTES

 
^* This work was supported by Deutsche Forschungsgemeinschaft Grant FR1487/3-1 and by funds from the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Ruhr-University Bochum, Physiology of Microorganisms, 44780 Bochum, Germany.

² A fellow of the Emmy-Noether program of the Deutsche Forschungsgemeinschaft. To whom the correspondence should be addressed: Ruhr-University Bochum, Physiology of Microorganisms, 44780 Bochum, Germany. Tel.: 49-234-32-23101; Fax: 49-234-32-14620; E-mail: nicole.frankenberg{at}rub.de.

³ The abbreviations used are: PEB, phycoerythrobilin; BV, biliverdin IX; DHBV, 15,16-dihydrobiliverdin; FDBR, ferredoxin-dependent bilin reductase; Fredi, Fremyella diplosiphon; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; PCB, phycocyanobilin; PcyA, phycocyanobilin:ferredoxin oxidoreductase; PE, phycoerythrin; PebA, 15,16-dihydrobiliverdin:ferredoxin oxidoreductase; PebB, phycoerythrobilin: ferredoxin oxidoreductase; PB, phytochromobilin; Synpy, Synechococcus sp. WH8020; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

	ACKNOWLEDGMENTS

 
We thank David Kehoe (Indiana University, Bloomington) for Fremyella diplosiphon genomic DNA and Donald Bryant (Pennsylvania State University, University Park) for plasmid pSe280Fd. We also thank Drs. J. C. Lagarias (University of California Davis) and M. Hollmann (Ruhr-University Bochum) for helpful discussions.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Alvey, R. M., Karty, J. A., Roos, E., Reilly, J. P., and Kehoe, D. M. (2003) Plant Cell 15, 2448–2463[Abstract/Free Full Text]
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