Insights into Phycoerythrobilin Biosynthesis Point toward Metabolic Channeling
2006; Elsevier BV; Volume: 281; Issue: 37 Linguagem: Inglês
10.1074/jbc.m605154200
ISSN1083-351X
AutoresThorben Dammeyer, Nicole Frankenberg‐Dinkel,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoPhycoerythrobilin 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 IXα, 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 IXα by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated π -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. 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 IXα, 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 IXα by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated π -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. 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). 3The abbreviations used are: PEB, phycoerythrobilin; BV, biliverdin IXα; 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; PΦB, phytochromobilin; Synpy, Synechococcus sp. WH8020; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.3The abbreviations used are: PEB, phycoerythrobilin; BV, biliverdin IXα; 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; PΦB, phytochromobilin; Synpy, Synechococcus sp. WH8020; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. 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 α- and β-subunits of PE (1Alvey R.M. Karty J.A. Roos E. Reilly J.P. Kehoe D.M. Plant Cell. 2003; 15: 2448-2463Crossref PubMed Scopus (37) Google Scholar). 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) (2Palenik B. Appl. Environ. Microbiol. 2001; 67: 991-994Crossref PubMed Scopus (164) Google Scholar), or, as recently suggested, by lyases that mediate PEB isomerization on the phycobiliproteins (3Everroad C. Six C. Partensky F. Thomas J.C. Holtzendorff J. Wood A.M. J. Bacteriol. 2006; 188: 3345-3356Crossref PubMed Scopus (90) Google Scholar). Because these organisms retain a fixed phycocyanin:PE ratio (4Waterbury J.B. Watson S.W. Valois F.W. Franks D.G. Can. Bull. Fish. Aquat. Sci. 1986; 214: 71-120Google Scholar), 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 IXα (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 (PΦB), 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 (5Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (5Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Evidence for the appearance of an intermediate substrate radical was recently demonstrated by absorbance and EPR spectroscopy (6Tu S.L. Gunn A. Toney M.D. Britt R.D. Lagarias J.C. J. Am. Chem. Soc. 2004; 126: 8682-8693Crossref PubMed Scopus (51) Google Scholar). Structural information to this new family of enzymes has recently been added through the solved crystal structure of the Synechocystis sp. PCC 6803 PcyA (7Hagiwara Y. Sugishima M. Takahashi Y. Fukuyama K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 27-32Crossref PubMed Scopus (52) Google Scholar). In contrast to the PcyA-catalyzed reaction the biosynthesis of PEB (which is an isomer of PCB) requires two independent enzymes, each catalyzing a two-electron reduction. 15,16-Dihydrobiliverdin:ferredoxin oxidoreductase (PebA) reduces the C-15 methine bridge of BV and phycoerythrobilin:ferredoxin oxidoreductase (PebB) the A-ring diene structure of 15,16-dihydrobiliverdin (DHBV), respectively. The biosynthesis of phycourobilin still remains unknown, but it might proceed analogously to the PecE/F isomerase/lyase activity of Mastigocladus laminosus, which covalently attaches and isomerizes PCB to yield bound phycoviolobilin (8Storf M. Parbel A. Meyer M. Strohmann B. Scheer H. Deng M.G. Zheng M. Zhou M. Zhao K.H. Biochemistry. 2001; 40: 12444-12456Crossref PubMed Scopus (74) Google Scholar). 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. 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™ DNA polymerase from Finnzymes. HPLC grade acetone, chloroform, and formic acid were purchased from Acros, Sigma-Aldrich, and J. T. Baker, respectively. Glutathione-Sepharose™ 4FF, PreScission™ 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™ accession number AY363679) were amplified from chromosomal DNA of F. diplosiphon (Fredi) strain Fd33 (9Cobley J.G. Zerweck E. Reyes R. Mody A. Seludo-Unson J.R. Jaeger H. Weerasuriya S. Navankasattusas S. Plasmid. 1993; 30: 90-105Crossref PubMed Scopus (78) Google Scholar) obtained from the laboratory of D. Kehoe. PCRs were set up using Phusion™ 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(λDE3) 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 (10Frankenberg N. Mukougawa K. Kohchi T. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (192) Google Scholar). 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(λDE3) carrying the respective plasmid construct and cultivated at 37 °C to an A578 nm ∼ 0.6–0.8. After a temperature shift to 17 °C, protein expression was induced by adding 100 μm isopropyl-β-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 × g the supernatant was loaded on a glutathione-Sepharose™ 4FF column. Washing and elution were done according to instructions supplied by the manufacturer. Protein-containing fractions were cleaved with 2 units of PreScission™ 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™ 26/60 Superdex™ 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 (11Schluchter W.M. The Characterization of Photosystem I and Ferredoxin-NADP+-Oxidoreductase in the Cyanobacterium Synechococcus sp. PCC 7002. Pennsylvania State University, 1994Google Scholar), and protein production was induced using 1 mm isopropyl-β-d-thiogalactopyranoside at an A578 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 (ϵ280 nm) (12Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5044) Google Scholar). The concentration of recombinantly produced ferredoxin from Synechococcus sp. PCC 7002 was determined using an ϵ420 nm of 9.7 mm–1 cm–1 (13Shin M. Oshino R. J. Biochem. 1978; 83: 357-361Crossref PubMed Scopus (78) Google Scholar). Concentration of BV IXα was calculated using an ϵ376 nm of 68.6 mm–1 cm–1 and an ϵ698 nm of 32.6 mm–1 cm–1 in 2.5% HCl-MeOH (14Heirwegh K.P. Blanckaert N. Van Hees G. Anal. Biochem. 1991; 195: 273-278Crossref PubMed Scopus (28) Google Scholar). The concentration of 3E-PEB was determined using ϵ326 nm 15.8 mm–1 cm–1 and ϵ591 nm 25.2 mm–1 cm–1 in 5% HCL-MeOH (15Cole W.J. Chapman D.J. Siegelman H.W. J. Am. Chem. Soc. 1967; 89: 5976-5977Crossref Scopus (69) Google Scholar). 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 (10Frankenberg N. Mukougawa K. Kohchi T. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (192) Google Scholar). 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 (16Clarke A.R. Engel P.C. Enzymology Labfax. Academic Press, London1995: 191-221Google Scholar). 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.F=Fmax×[enzyme](Kd+[enzyme])(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).FIGURE 5Spectroscopic 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 Large Image Figure ViewerDownload Hi-res image Download (PPT) HPLC-Analysis—Bilin reaction products were analyzed as described previously (5Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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™-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). 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. Activity of the Recombinant Enzymes—To verify the activity of the purified bilin reductases, we used an in vitro assay system as described previously with an excess of reductant (10Frankenberg N. Mukougawa K. Kohchi T. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (192) Google Scholar). The optimal pH value for PebAFredi activity was determined to be pH 7.5, and therefore all further assays were performed at this pH. As expected, both PebAs were found to convert BV to DHBV (Fig. 3). The reduction of the 15,16-double bond of BV is accompanied by obvious blue-shifts of the absorption that enabled us to monitor the in vitro reaction progress spectroscopically (shown in Fig. 4). The analyzed PebBs catalyzed the reduction of the A-ring diene system of DHBV to PEB, which may likely be a 2,3-reduction, followed by isomerization to 3Z-PEB, which is the supposed natural chromophore of PE. The overall reaction can be followed by HPLC in an assay mixture containing BV, PebA, and PebB at once (Fig. 3) or individually using BV as a substrate for PebA or purified DHBV as substrate for PebB (data not shown) to confirm the specific catalytic activity for both enzymes. The appearance of the energetically stable 3E-PEB may be a result of the bilin extraction procedure as described previously (10Frankenberg N. Mukougawa K. Kohchi T. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (192) Google Scholar). In our assay system both reactions were found to be most efficient using plant type [2Fe-2S] ferredoxins of Synechococcus sp. PCC7002 or S. oleacera as redox partners followed by the [2Fe-2S] ferredoxin from P. umbilicalis; only marginal activity could be detected using [4Fe-4S] ferredoxin from C. pasteurianum. These results are in good agreement with results obtained for PcyA of Anabaena sp. PCC7120 (5Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and for phytochromobilin synthase of Avena sativa (17McDowell M.T. Lagarias J.C. Plant Physiol. 2001; 126: 1546-1554Crossref PubMed Scopus (18) Google Scholar). Consistent with PcyA is the insensitivity of PebAFredi toward metal chelators like EDTA (10 μm), o-phenanthroline (5 μm), or 2,2′-dipyridyl (5 μm), indicating no involvement of protein-associated metal ion cofactors during catalysis (data not shown) (5Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Reoxidation of 15,16-DHBV—The PebA-catalyzed reduction of BV to DHBV was found to be reversible. Incubation of a preformed complex of PebA and DHBV for 3–6 days on ice in the dark led to a visible transformation of the color from pink to green, the result of a back-oxidation of DHBV to BV, which was confirmed by HPLC (data not shown). This back-oxidation was slower under low oxygen conditions; a control experiment with carbonic anhydrase instead of PebA in the solution resulted in much lower DHBV reoxidation, indicating that this reaction is accelerated in the presence of PebA. 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 PΦB to PEB, indicating that an A-ring ethylidene group instead of an A-ring vinyl group is not critical for substrate recognition by PebA.TABLE 1Substrate specificity of FDBRs from cyanobacteriaBilin substrateFDBRPebAPebBPcyABV IXαDHBVNMaNM, not metabolized.3E-/3Z-PCBDHBVNM3E-/3Z-PEBNM3E-PCBNMNMNM3E-/3Z-PΦB3E-/3Z-PEBbThe substrate used in the assay was the product of the HY2 reaction, which yields the 3E- and 3Z-isomers of PΦB.NMNM/3E-/3Z-PCBcOnly 3Z-PΦB is metabolized (5).181182-DHBVNDdND, not determined.NM3E-/3Z-PCBa NM, not metabolized.b The substrate used in the assay was the product of the HY2 reaction, which yields the 3E- and 3Z-isomers of PΦB.c Only 3Z-PΦB is metabolized (5Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).d ND, not determined. Open table in a new tab Spectroscopic Properties of FDBR Complexes with Their Substrates or Products—All tested bilins incubated with PebA or PebB displayed distinct spectroscopic properties differing from those of the free pigments (Fig. 5). Not only were the absorbance maxima shifted, but also the peak intensities and the ratio of the long wavelength absorbance peak and the near UV absorbance peak intensities (λmax2/λmax1) changed (Table 2). Interestingly, the protein environment of PebA and PebB influences the spectral properties of one and the same bilin, indicating differences in the bilin binding pocket of both FDBRs. BV binding to PebA causes an increase in absorbance compared with free BV, with a shifted long wavelength absorbance maximum from 681 to 691 nm. Bound to PebB, the absorbance maximum is less intensely increased, but the long wavelength absorbance maximum is shifted from 681 to 706 nm. Consequently, the ratio of λmax2/λmax1 did not change notably, and lies between 0.3 for BV and 0.52 for PebA·BV (Fig. 5A and Table 2). Spectral analyses of complexes of the semi-reduced intermediate DHBV with PebA (enzyme·product complex) and PebB (enzyme·substrate complex) revealed noticeable differences. DHBV binding to PebA led to an absorbance increase at both maxima and to a shift from 565 to 590 nm for λmax2. Binding to PebB shifted the λmax2 to 605 nm and decreased the λmax1 absorbance, thereby changing the color of the complex from pink to blue. The λmax2/λmax1 ratio was changed from 0.63 (free DHBV) to 0.97 (PebA·DHBV) and 1.36 (PebB·DHBV) (Fig. 5B and Table 2). The binding of 3E-PEB to PebB led to an increased absorbance at the long wavelength absorbance
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