Biosynthesis Of Phycocyanobilin Research Articles

Rational Design of Key Enzymes to Efficiently Synthesize Phycocyanobilin in Escherichia coli.

Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from Spirulina involves complex processes, which has led to increasing interest in the biosynthesis of PCB in Escherichia coli. However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from Thermosynechococcus elongatus BP-1(HOT) and PCB: ferredoxin oxidoreductase from Synechocystis sp. PCC6803 (PcyAS). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HOT(F29W/K166D) and PcyAS(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HOT and PcyAS mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of hemB and hemH, resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.

On the evolution of the plant phytochrome chromophore biosynthesis.

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins

Terrestrial ecosystems and human societies depend on oxygenic photosynthesis, which began to reshape our atmosphere approximately 2.5 billion years ago. The earliest known organisms carrying out oxygenic photosynthesis are the cyanobacteria, which use large complexes of phycobiliproteins as light-harvesting antennae. Phycobiliproteins rely on phycocyanobilin (PCB), a linear tetrapyrrole (bilin) chromophore, as the light-harvesting pigment that transfers absorbed light energy from phycobilisomes to the chlorophyll-based photosynthetic apparatus. Cyanobacteria synthesize PCB from heme in two steps: A heme oxygenase converts heme into biliverdin IXα (BV), and the ferredoxin-dependent bilin reductase (FDBR) PcyA then converts BV into PCB. In the current work, we examine the origins of this pathway. We demonstrate that PcyA evolved from pre-PcyA proteins found in nonphotosynthetic bacteria and that pre-PcyA enzymes are active FDBRs that do not yield PCB. Pre-PcyA genes are associated with two gene clusters. Both clusters encode bilin-binding globin proteins, phycobiliprotein paralogs that we designate as BBAGs (bilin biosynthesis-associated globins). Some cyanobacteria also contain one such gene cluster, including a BBAG, two V4R proteins, and an iron-sulfur protein. Phylogenetic analysis shows that this cluster is descended from those associated with pre-PcyA proteins and that light-harvesting phycobiliproteins are also descended from BBAGs found in other bacteria. We propose that PcyA and phycobiliproteins originated in heterotrophic, nonphotosynthetic bacteria and were subsequently acquired by cyanobacteria.

Enhancement of phycocyanobilin biosynthesis in Escherichia coli by strengthening the supply of precursor and artificially self-assembly complex

Phycocyanobilin (PCB) is widely used in healthcare, food processing, and cosmetics. Escherichia coli is the common engineered bacterium used to produce PCB. However, it still suffers from low production level, precursor deficiency, and low catalytic efficiency. In this study, a highly efficient PCB-producing strain was created. First, chassis strains and enzyme sources were screened, and copy numbers were optimized, affording a PCB titer of 9.1 mg/L. Most importantly, the rate-limiting steps of the PCB biosynthetic pathway were determined, and the supply of precursors necessary for PCB synthesis was increased from endogenous sources, affording a titer of 21.4 mg/L. Then, the key enzymes for PCB synthesis, HO1 and PcyA, were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD, and 23.5 mg/L of PCB was obtained. Finally, the basic conditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB. The final titer of PCB reached 147.0 mg/L, which is the highest reported titer of PCB so far. This research provided the foundation for the industrial production of PCB and its derivatives.

Efficient Synthesis of Phycocyanobilin by Combinatorial Metabolic Engineering in Escherichia coli.

Phycocyanobilin (PCB) is a kind of light-harvesting pigment which naturally exists in algae and plays important roles in absorbing and transferring energy. Based on its antioxidant and optical properties, PCB has been applied in food, medicine, and cosmetics. Currently, PCB is mainly extracted from Spirulina through complicated steps; thus, the biosynthesis of PCB in Escherichia coli has attracted more attention. However, due to the lower catalytic efficiency of synthetic enzymes and the deficiency of precursors and cofactors, the titer of PCB remains at a low level. Here, we report the efficient synthesis of PCB by the expression of heme oxygenase-1 from Thermosynechococcus elongatus and PCB: ferredoxin oxidoreductase (PcyA) from Synechocystis sp. using a high-copy number plasmid with an inducible T7lac promoter and the assembly of these two enzymes at a suitable ratio of 2:1 with DNA scaffolds. Additionally, the synthesis of PCB was further enhanced by direct supplementation of 5-aminolevulinic acid (ALA), moderate overexpression of key enzymes in the heme biosynthetic pathway (hemB and hemH), and accelerated cycle of cofactors (NADPH) through the expression of NAD+ kinase and the addition of a reducing agent. Finally, based on the optimal conditions (Modified R medium with 200 mg/L ALA, 20 mg/L FeSO4·7H2O, and 5 g/L vitamin C induced by 0.8 mM isopropylthio-β-galactoside at 30 °C), the highest reported titer of PCB (28.32 mg/L) was obtained at the fermenter level by feeding glucose and FeSO4·7H2O. The strategies applied in this study will be useful for the synthesis of other natural pigments and PCB or heme derivatives in E. coli.

Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system.

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV both in vitro and in fission yeast. We introduced SynPCB2.1, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB2.1. These tools not only enable the easy use of multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms. This article has an associated First Person interview with the first author of the paper.

First person – Keiichiro Sakai

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Keiichiro Sakai is first author on ‘ Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system’, published in JCS. Keiichiro is a PhD student in the lab of Kazuhiro Aoki at the Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Aichi, Japan, investigating how phycocyanobilin, a linear tetrapyrrole, brightens near-infrared fluorescent proteins, including iRFP, as a chromophore more efficiently than biliverdin in fission yeast.

Nitrogen Sources and Iron Availability Affect Pigment Biosynthesis and Nutrient Consumption in Anabaena sp. UTEX 2576.

Anabaena sp. UTEX 2576 metabolizes multiple nitrogen (N) sources and is deemed a biotechnological platform for chemical production. Cyanobacteria have been identified as prolific producers of biofertilizers, biopolymers, biofuels, and other bioactive compounds. Here, we analyze the effect of different N-sources and Fe availability on the bioproduction of phycobiliproteins and β-carotene. We characterize nutrient demand in modified BG11 media, including data on CO2 fixation rates, N-source consumption, and mineral utilization (e.g., phosphorus (P), and 11 metallic elements). Results suggest that non-diazotrophic cultures grow up to 60% faster than diazotrophic cells, resulting in 20% higher CO2-fixation rates. While the production of β-carotene was maximum in medium with NaNO3, Fe starvation increased the cellular abundance of C-phycocyanin and allophycocyanin by at least 22%. Compared to cells metabolizing NaNO3 and N2, cultures adapted to urea media increased their P, calcium and manganese demands by at least 72%, 97% and 76%, respectively. Variations on pigmentation and nutrient uptake were attributed to changes in phycocyanobilin biosynthesis, light-induced oxidation of carotenoids, and urea-promoted peroxidation. This work presents insights into developing optimal Anabaena culture for efficient operations of bioproduction and wastewater bioremediation with cyanobacteria.

Regulation of the heme biosynthetic pathway for combinational biosynthesis of phycocyanobilin in Escherichia coli

Abstract Due to its biochemical and spectroscopic properties, phycocyanobilin (PCB) is of special interest for a variety of applications, but the production of PCB from Spirulina is complicated and costly. Here we report the assembly and regulation of the entire heme biosynthetic pathway for combinatorial biosynthesis of PCB in Escherichia coli. Our results shows that a plasmid containing bicistronic expression of hox1 and pcyA yields 6.64 mg/L PCB, which is higher than that of plasmid with one promoter for both genes (p

Photochemical Properties of a Novel Cyanobacteriochrome from Anabaena sp. PCC7120

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as photoreceptor sensors. We report the photochemistry of All4261 GAF2, a novel cyanobacteriochrome from the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. All4261 contains four tandemly arranged GAF domains. The respective chromophore domains were co-expressed in Escherichia coli with the genes for phycocyanobilin biosynthesis enzymes, HO1, and PcyA. The resulting proteins were analyzed by zinc-induced fluorescence, UV-Vis absorption and emission fluorescence spectra. Only All4261 GAF2 binded chromophore covalently, having zinc-induced fluorescence band in SDS-PAGE gel with the zinc acetate solution existed. Absorption spectra analysis showed that All4261 GAF2 had absorption peaks at 340 nm and 590 nm, failing to show photoreversibility. Fluorescence spectra revealed that All4261 GAF2 had a fluorescence emission peak at 645 nm, with high fluorescence quantum yield and molar extinction coefficient.

Combinational biosynthesis of phycocyanobilin using genetically-engineered Escherichia coli

Genes of the key enzymes for phycocyanobilin (PCB) biosynthesis were cloned into E. coli and combinationally expressed to produce phycocyanobilin, with autologous heme as substrate. Culture conditions were optimized to achieve ~3 mg PCB/l. A protocol for the purification of recombinant phycocyanobilin was established using solvent extraction combined with chromatography, which resulted in a final yield of ~0.3 mg PCB/l with a purity >95 %. Recombinant phycocyanobilin could scavenge hydroxyl radicals with an EC50 of 0.1 μM.

A rising tide of blue‐absorbing biliprotein photoreceptors – characterization of seven such bilin‐binding GAF domains in Nostoc sp. PCC7120

Cyanobacteriochromes are photochromic sensory photoreceptors in cyanobacteria that are related to phytochromes but cover a much broader spectral range. Using a homology search, a group of putative blue-absorbing photoreceptors was identified in Nostoc sp. PCC 7120 that, in addition to the canonical chromophore-binding cysteine of cyanobacteriochromes, have a conserved extra cysteine in a DXCF motif. To assess their photochemical activities, putative chromophore-binding GAF domains were expressed in Escherichiacoli together with the genes for phycocyanobilin biosynthesis. All except one covalently bound a chromophore and showed photoreversible photochromic responses, with absorption at approximately 420nm for the 15Z states formed in the dark, and a variety of red-shifted absorption peaks in the 490-600nm range for the 15E states formed after light activation. Under denaturing conditions, the covalently bound chromophores were identified as phycocyanobilin, phycoviolobilin or mixtures of both. The canonical cysteines and those of the DXCF motifs were mutated, singly or together. The canonical cysteine is responsible for stable covalent attachment of the bilin to the apo-protein at C3(1) . The second linkage from the cysteine in the DXCF motif, probably to C10 of the chromophore, yields blue-absorbing rubin-type 15Z chromophores, but is lost in most cases upon photoconversion to the 15E isomers of the chromophores, and also when denatured with acidic urea.

Molecular characterization of a novel gene from Synechocystis sp. PCC 6803

A novel gene slr2049 was identified in Synechococcus sp . PCC7002 by homologous alignment. The features and possible functions of slr2049 gene were predicted by bioinformatics analysis. The function of slr2049 was analyzed in vitro with a heterologous Escherichia coli system with plasmids conferring biosynthesis of phycocyanobilin (PCB) and of the acceptor proteins, β-phycocyanin (CpcB). The resulting products were evaluated with SDS-PAGE and absorption spectra. The function of slr2049 was further analyzed via site-directed mutations. Two mutants, slr2049 (W14L) and slr2049 (Y132S) were generated. The results showed that Slr2049 could catalyze the chromophorylation of CpcB. Compared to wild type, mutant Slr2049 (W14L) had red-shifted absorbance maxima and was not highly fluorescent as the wild-type. However, mutant Slr2049 (Y132S) was almost the same as the wild-type. In conclusion, our study suggests that we have cloned a novel gene and this gene may play an important role in attachment of the chromophores to the apo-proteins.

Effects of Modified Phycobilin Biosynthesis in the CyanobacteriumSynechococcussp. Strain PCC 7002

The pathway for phycocyanobilin biosynthesis in Synechococcus sp. strain PCC 7002 comprises two enzymes: heme oxygenase and phycocyanobilin synthase (PcyA). The phycobilin content of cells can be modified by overexpressing genes encoding alternative enzymes for biliverdin reduction. Overexpression of the pebAB and HY2 genes, encoding alternative ferredoxin-dependent biliverdin reductases, caused unique effects due to the overproduction of phycoerythrobilin and phytochromobilin, respectively. Colonies overexpressing pebAB became reddish brown and visually resembled strains that naturally produce phycoerythrin. This was almost exclusively due to the replacement of phycocyanobilin by phycoerythrobilin on the phycocyanin α-subunit. This phenotype was unstable, and such strains rapidly reverted to the wild-type appearance, presumably due to strong selective pressure to inactivate pebAB expression. Overproduction of phytochromobilin, synthesized by the Arabidopsis thaliana HY2 product, was tolerated much better. Cells overexpressing HY2 were only slightly less pigmented and blue-green than the wild type. Although the pcyA gene could not be inactivated in the wild type, pcyA was easily inactivated when cells expressed HY2. These results indicate that phytochromobilin can functionally substitute for phycocyanobilin in Synechococcus sp. strain PCC 7002. Although functional phycobilisomes were assembled in this strain, the overall phycobiliprotein content of cells was lower, the efficiency of energy transfer by these phycobilisomes was lower than for wild-type phycobilisomes, and the absorption cross-section of the cells was reduced relative to that of the wild type because of an increased spectral overlap of the modified phycobiliproteins with chlorophyll a. As a result, the strain producing phycobiliproteins carrying phytochromobilin grew much more slowly at low light intensity.

A light regulated OmpR‐class promoter element co‐ordinates light‐harvesting protein and chromophore biosynthetic enzyme gene expression

Co-ordination of chromophore and apoprotein biosynthesis is required during photosynthetic light-harvesting antennae production, such as occurs during complementary chromatic adaptation (CCA). This response to ambient light colour changes is controlled by a phytochrome-class photoreceptor and involves changes in the synthesis of cyanobacterial light-harvesting antennae. During growth in red light, CCA activates cpc2 transcription, an operon that encodes the light-harvesting protein phycocyanin. In order to function, this apoprotein must have covalently attached phycocyanobilin chromophores, which are synthesized by PcyA. We show that pcyA is also transcriptionally activated by CCA during red light growth and is not regulated via feedback that senses cpc2 RNA levels. The pcyA and cpc2 promoters contain a common regulatory element, a direct repeat typical of OmpR-class transcription factor binding sites, at similar positions relative to their red light-controlled transcription start sites. Deletion of this element from the pcyA promoter eliminated CCA-regulated transcription, and insertion of the element into a non-light responsive promoter conferred CCA regulation. We conclude that this element is necessary and sufficient to confer CCA transcriptional regulation and that it co-ordinates phycocyanin and phycocyanobilin biosynthesis in red light.

Insights into Phycoerythrobilin Biosynthesis Point toward 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 IXalpha, 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 IXalpha by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated pi -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3(1),3(2)-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.

Crystal structure of phycocyanobilin:ferredoxin oxidoreductase in complex with biliverdin IXalpha, a key enzyme in the biosynthesis of phycocyanobilin.

Phytobilins (light harvesting and photoreceptor pigments in higher plants, algae, and cyanobacteria) are synthesized from biliverdin IXalpha (BV) by ferredoxin-dependent bilin reductases (FDBRs). Phycocyanobilin:ferredoxin oxidoreductase (PcyA), one such FDBR, is a new class of radical enzymes that require neither cofactors nor metals and serially reduces the vinyl group of the D-ring and A-ring of BV using four electrons from ferredoxin to produce phycocyanobilin, one of the phytobilins. We have determined the crystal structure of PcyA from Synechocystis sp. PCC 6803 in complex with BV, revealing the first tertiary structure of an FDBR family member. PcyA is folded in a three-layer alpha/beta/alpha sandwich structure, in which BV in a cyclic conformation is positioned between the beta-sheet and C-terminal alpha-helices. The basic patch on the PcyA surface near the BV molecule may provide a binding site for acidic ferredoxin, allowing direct transfer of electrons to BV. The orientation of BV is definitely fixed in PcyA by several hydrophilic interactions and the shape of the BV binding pocket of PcyA. We propose the mechanism by which the sequential reduction of the D- and A-rings is controlled, where Asp-105, located between the two reduction sites, would play the central role by changing its conformation during the reaction. Homology modeling of other FDBRs based on the PcyA structure fits well with previous genetic and biochemical data, thereby providing a structural basis for the reaction mechanism of FDBRs.

Inhibition by glucose of chlorophyll a and phycocyanobilin biosynthesis in the unicellular red alga Galdieria partita at the stage of coproporphyrinogen III formation

The repression effect of d-glucose on the pigment apparatus of photosynthesis in the unicellular acidophilic red alga Galdieria partita capable of heterotrophic growth was studied. The rate of cell growth under heterotrophic conditions was higher by more than one order of magnitude than that in the autotrophic ones, and the size of those cells was larger. The addition of 1% d-glucose to the growth medium caused an increase in the number of mitochondrion profiles in the cell and a decline of thylakoid number in the chloroplast of G. partita. The loss of thylakoid membranes is accompanied by a reduction of the contents of chlorophyll a. C-phycocyanin and allophycocyanin in the heterotrophic cells. Simultaneously, switching G. partita from autotrophic growth to heterotrophic nutrition led to the excretion of coproporphyrin(ogen) III into the growth medium. Thus, the presence of d-glucose caused a reduction of pigment contents in the cell, due to the inhibition of chlorophyll a and phycocyanobilin biosynthesis at the stage of the transformation of their universal precursor, coproporphyrinogen III, to protoporphyrinogen IX. In the mixotrophic culture of G. partita light induces both the biosynthesis of photosynthetic pigments and the excretion of coproporphyrin(ogen) III. It is supposed that light affects an earlier stage in the biosynthetic pathway of chlorophyll a and phycocyanobilin than does d-glucose.

The biosynthesis of the chromophore of phycocyanin. Pathway of reduction of biliverdin to phycocyanobilin

The later stages in the pathway of biosynthesis of phycocyanobilin, the chromophore of phycocyanin, were studied by using radiolabelled intermediates. Three possible pathways from biliverdin IX-alpha to phycocyanobilin were considered. 14C-labelled samples of key intermediates in two of the pathways, 3-vinyl-18-ethyl biliverdin IX-alpha and 3-ethyl-18-vinyl biliverdin IX-alpha, were synthesized chemically and were administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Neither of these two compounds was apparently incorporated into the phycobiliprotein chromophore, suggesting that two of the three pathways were not operative. By elimination, the results imply that the third possible pathway, which involves phytochromobilin, the chromophore of phytochrome, represents the route for biosynthesis of phycocyanobilin. Unfortunately, since 14C-labelled phytochromobilin is not available, no direct proof of this pathway could be obtained. However, if correct, the present interpretation represents a unified pathway for biosynthesis of all plant bilins, via the intermediacy of phytochromobilin.

Biosynthesis of phycocyanobilin from exogenous labeled biliverdin in Cyanidium caldarium

Phycocyanin is a major light-harvesting pigment in bluegreen, red, and cryptomonad algae. This pigment is composed of phycocyanobilin chromophores covalently attached to protein. Phycocyanobilin is an open-chain tetrapyrrole structurally close to biliverdin. Biliverdin is formed in animals by oxidative ring-opening of protoheme. Recent evidence indicates that protoheme is a precursor of phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium. To find out if biliverdin is an intermediate in the conversion of protoheme to phycocyanobilin, [ 14C]biliverdin was administered along with N-methylmesoporphyrin IX (which blocks endogenous protoheme formation) to growing cells of C. caldarium. To avoid phototoxic effects due to the porphyrin, a mutant strain was used that forms large amounts of both chlorophyll and phycocyanin in the dark. After 12 or 24 h in the dark, cells were harvested and exhaustively extracted to remove free pigments. Next, protoheme was extracted. Phycocyanobilin was then cleaved from the apoprotein by methanolysis. Protoheme and phycocyanobilin were purified by solvent partition, DEAE-Sepharose chromatography, and preparative reverse-phase highpressure liquid chromatography. Absorption was monitored continuously and fractions were collected for radioactivity determination. Negligible amounts of label appeared in the protoheme-containing fractions. A major portion of label in the eluates of the phycocyanobilin-containing samples coincided with the absorption peak at 22 min due to phycocyanobilin. In a control experiment, [ 14C]biliverdin was added to the cells after incubation and just before the phycocyanobilin-apoprotein cleavage step. The major peak of label then eluted with the absorption peak at 12 min due to biliverdin, indicating that during the isolation biliverdin is not converted to compounds coeluting with phycocyanobilin. It thus appears that exogenous biliverdin can serve as a precursor to phycocyanobilin in C. caldarium, and that the route of incorporation is direct rather than by degradation and reincorporation of 14C through protoheme.