Biosynthesis of phycobilins. 3(Z)-phycoerythrobilin and 3(Z)-phycocyanobilin are intermediates in the formation of 3(E)-phycocyanobilin from biliverdin IX alpha

An enzyme extract from the phycocyanin-containing unicellular rhodophyte, Cyanidium caldarium, reductively transforms biliverdin IX alpha to phycocyanobilin, the chromophore of phycocyanin, in the presence of NADPH. Unpurified cell extract forms both 3(E)-phycocyanobilin, which is identical to the major pigment that is released from phycocyanin by methanolysis, and 3(Z)-phycocyanobilin, which is obtained as a minor methanolysis product. After removal of low molecular weight material from the cell extract, only 3(Z)-phycocyanobilin is formed. 3(E)-Phycocyanobilin formation from biliverdin IX alpha, and the ability to isomerize 3(Z)-phycocyanobilin to 3(E)-phycocyanobilin, are reconstituted by the addition of glutathione to the incubation mixture. Partially purified protein fractions derived from the initial enzyme extract form 3(Z)-phycocyanobilin plus two additional, violet colored bilins, upon incubation with NADPH and biliverdin IX alpha. Further purified protein fractions produce only the violet colored bilins from biliverdin IX alpha. One of these bilins was identified as 3(Z)-phycoerythrobilin by comparative spectrophotometry, reverse-phase high pressure liquid chromatography, and 1H NMR spectroscopy. A C. caldarium protein fraction catalyzes the conversion of 3(Z)-phycoerythrobilin to 3(Z)-phycocyanobilin. This fraction also catalyzes the conversion of 3(E)-phycoerythrobilin to 3(E)-phycocyanobilin. The conversion of phycoerythrobilins to phycocyanobilins requires neither biliverdin nor NADPH. The synthesis of phycoerythrobilin and its conversion to phycocyanobilin by extracts of C. caldarium, a species that does not contain phycoerythrin, indicates that phycoerythrobilin is a biosynthetic precursor to phycocyanobilin. The enzymatic conversion of the ethylidine group from the Z to the E configuration suggests that the E-isomer is the precursor to the protein-bound chromophore.     

Biosynthesis of the chromophore of phycobiliproteins. A study of mesohaem and mesobiliverdin as possible intermediates and further evidence for an algal haem oxygenase.

The possible roles of mesohaem and mesobiliverdin as metabolic precursors of phycocyanobilin, the chromophore of phycocyanin, were studied in the unicellular rhodophyte Cyanidium caldarium. Dark-grown cells of this organism, which had been exposed to mesohaem, were either incubated in the dark with 5-aminolaevulinate, which results in excretion of bilins into the suspending medium, or incubated in the light, which results in synthesis of phycocyanin within the cells. By using 14C-labelling, either in the mesohaem or in the 5-aminolaevulinate administered, it was shown that mesohaem is not a precursor of phycocyanobilin in either dark or light systems. However, mesohaem was converted into mesobiliverdin in both systems, a phenomenon that is further evidence for the existence of an algal haem oxygenase. The data also showed that mesobiliverdin is not a precursor of phycocyanobilin. These results suggest that algal bilins are formed via haem degradation to biliverdin in the same way as mammalian bile pigments.     

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 high-pressure 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.     

Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin beta-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin beta-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin beta-subunit.     

Chromophore composition of the phycobiliprotein Cr-PC577 from the cryptophyte Hemiselmis pacifica

The cryptophyte phycocyanin Cr-PC577 from Hemiselmis pacifica is a close relative of Cr-PC612 found in Hemiselmis virescens and Hemiselmis tepida. The two biliproteins differ in that Cr-PC577 lacks the major peak at around 612 nm in the absorption spectrum. Cr-PC577 was thus purified and characterized with respect to its bilin chromophore composition. Like other cryptophyte phycobiliproteins, Cr-PC577 is an (αβ)(α′β) heterodimer with phycocyanobilin (PCB) bound to the α-subunits. While one chromophore of the β-subunit is also PCB, mass spectrometry identified an additional chromophore with a mass of 585 Da at position β-Cys-158. This mass can be attributed to either a dihydrobiliverdin (DHBV), mesobiliverdin (MBV), or bilin584 chromophore. The doubly linked bilin at position β-Cys-50 and β-Cys-61 could not be identified unequivocally but shares spectral features with DHBV. We found that Cr-PC577 possesses a novel chromophore composition with at least two different chromophores bound to the β-subunit. Overall, our data contribute to a better understanding of cryptophyte phycobiliproteins and furthermore raise the question on the biosynthetic pathway of cryptophyte chromophores.     

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 phycobilins. Ferredoxin-mediated reduction of biliverdin catalyzed by extracts of Cyanidium caldarium

Cell-free extract of the unicellular rhodophyte, Cyanidium caldarium catalyzes enzymatic reduction of biliverdin IX alpha to phycocyanobilin, the chromophore of the light-harvesting phycobiliprotein, phycocyanin. The enzyme activity is soluble, and the required reductant is NADPH. The extract has been separated into three protein fractions, all of which are required to reconstitute biliverdin reduction. One fraction contains ferredoxin, which was identified by its absorption spectrum. This fraction could be replaced with commercial ferredoxin derived from spinach or the red alga, Porphyra umbilicalis. The second protein fraction contains ferredoxin-NADP+ reductase, which was identified by the ability to catalyze ferredoxin-dependent reduction of cytochrome c in the presence of NADPH. This fraction could be replaced with commercial spinach ferredoxin-NADP+ reductase. These two components appear to be identical to previously described components of the algal heme oxygenase system that catalyzes biliverdin IX alpha formation from protoheme in C. caldarium extracts. The third protein fraction, in the presence of the first two (or their commercial counterparts) plus NADPH, catalyzes the reduction of biliverdin IX alpha to phycocyanobilin. The results indicate that the transformation of biliverdin to phycocyanobilin catalyzed by C. caldarium extracts is a ferredoxin-linked reduction process. The results also suggest the possibility that heme oxygenation and biliverdin reduction may occur in C. caldarium on associated enzyme systems.     

Studies on the Formation of Phycocyanin, Porphyrins, and a Blue Phycobilin by Wild-Type and Mutant Strains of Cyanidium caldarium

The synthesis of chlorophyll a and the bile-pigment and protein moieties of phycocyanin were arrested in illuminated cells of Cyanidium caldarium, strain III-D-2, incubated with chloramphenicol, ethionine, p-fluorophenylalanine, and p-chloromercuribenzoate. Pigment synthesis was similarly retarded in illuminated cells provided with nutrient medium lacking nitrogen.

Porphobilinogen, porphyrins, and a blue phycobilin were excreted into the nutrient medium by illuminated and unilluminated cells of wild-type and mutant C. caldarium strains incubated with δ-aminolevulinic acid in darkness. Pigment production from δ-aminolevulinic acid was sensitive to treatment with chloramphenicol and ethionine.

Cells of C. caldarium excreted 7 red-fluorescing porphyrins into the suspending medium during incubation with δ-aminolevulinic acid. Three of these porphyrins were identified as uroporphyrin III, coproporphyrin III, and protoporphyrin on the basis of their spectral properties and by paper chromatogaphy with standards.

The blue phycobilin was characterized spectrally and compared with biliverdin. The algal phycobilin displayed properties of a pigment with a violin-type structure. The phycobilin may be an immediate precursor of phycocyanobilin, the phycocyanin chromophore, or identical to it.

     

Characterization of the acetyltransferase used in pheromone biosynthesis in moths: Specificity for the Z isomer in tortricidae

Characterization of the acetyltransferase (acetyl-CoA: fatty alcohol acetyltransferase) that catalyzes the conversion of fatty alcohols to acetate esters was carried out in the redbanded leafroller moth, Argyrotaenia velutinana. This enzyme is one of the last enzymes used in the pheromone biosynthetic pathway of many moths. Characterization of the acetyltransferase involved an in vitro enzyme assay using a pheromone gland homogenate. The enzyme found only in the pheromone gland, was active during the photophase and at all adult ages tested (1–5-days-old). The enzyme was derermined to be microsomal and exhibited specificity for the Z isomer of all 12, 14 and 16 carbon monounsaturated fatty alcohol substrates tested. The shape of the kinetic curves for the Z and E isomers of Δ11-tetradecenol were similar for time of incubation, protein and substrate concentration, with the Z isomer always forming 1.5–2 times more product. Substrate competition assays between Z and E11-tetradecenol indicate that one enzyme is involved and it converts the Z isomer to product faster than the E isomer. Substrate preference assays conducted with the enzyme from Choristoneura rosaceana, C. fumiferana and Platynota idaeusalis (all Tortricidae) pheromone glands also indicate specificity for the Z isomer. On the other hand, assays conducted with Trichoplusia ni (Noctuidae) and three strains (Z, E and hybrid) of Ostrinia nubilalis (Pyralidae) indicate no preferences between the Z and E isomers.     

CpeS Is a Lyase Specific for Attachment of 3Z-PEB to Cys82 of β-phycoerythrin from Prochlorococcus marinus MED4

In contrast to the majority of cyanobacteria, the unicellular marine cyanobacterium Prochlorococcus marinus MED4 uses an intrinsic divinyl-chlorophyll-dependent light-harvesting system for photosynthesis. Despite the absence of phycobilisomes, this high-light adapted strain possesses β-phycoerythrin (CpeB), an S-type lyase (CpeS), and enzymes for the biosynthesis of phycoerythrobilin (PEB) and phycocyanobilin. Of all linear tetrapyrroles synthesized by Prochlorococcus including their 3Z- and 3E-isomers, CpeS binds both isomers of PEB and its biosynthetic precursor 15,16-dihydrobiliverdin (DHBV). However, dimerization of CpeS is independent of bilins, which are tightly bound in a complex at a ratio of 1:1. Although bilin binding by CpeS is fast, transfer to CpeB is rather slow. CpeS is able to attach 3E-PEB and 3Z-PEB to dimeric CpeB but not DHBV. CpeS transfer of 3Z-PEB exclusively yields correctly bound βCys⁸²-PEB, whereas βCys⁸²-DHBV is a side product of 3E-PEB transfer. Spontaneous 3E- and 3Z-PEB addition to CpeB is faulty, and products are in both cases βCys⁸²-DHBV and likely a PEB bound at βCys⁸² in a non-native configuration. Our data indicate that CpeS is specific for 3Z-PEB transfer to βCys⁸² of phycoerythrin and essential for the correct configuration of the attachment product.     

Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic

In cyanobacteria, the biosynthesis of the phycobiliprotein and phytochrome chromophore precursor phycocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduction of biliverdin IXalpha. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native M(r) of 30,400 +/- 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IXalpha. The enzyme exhibits a strong preference for plant type [2Fe-2S] ferredoxins; however, flavodoxin can also serve as an electron donor. HPLC analyses establish that catalysis proceeds via the two electron-reduced intermediate 18(1),18(2)-dihydrobiliverdin, indicating that exovinyl reduction precedes A-ring (endovinyl) reduction. Substrate specificity studies indicate that the arrangement of the A- and D-ring substituents alters the positioning of the bilin substrate within the enzyme, profoundly influencing the course of catalysis. Based on these observations and the apparent lack of a metal or small molecule cofactor, a radical mechanism for biliverdin IXalpha reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.     

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.     

Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin‐dependent heme oxygenase from Synechocystis sp. PCC 6803

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXα, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IXα. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IXα by spectrophotometry and reverse-phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100 000 g. Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.     

Molecular cloning and expression analysis of a new bilin lyase: the cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314

To study the assembly of phycocyanin β subunit, the gene cpcT was first cloned from Arthrospira platensis FACHB314. To explore the function of cpcT, the DNA of phycocyanin β subunit and cpcT were transformed into Escherichia coli BL21 with the plasmid pET-hox1-pcyA, which contained the genes hemeoxygenase 1 (Hox1) and ferredoxin oxidoreductase (PcyA) needed to produce phycocyanobilin. The transformed strains showed specific phycocyanin fluorescence, and the fluorescence intensity was stronger than the strains with only phycocyanin β subunit, indicating that CpcT can promote the assembly of phycocyanin to generate fluorescence. To study the possible binding sites of apo-phycocyanin and phycocyanobilin, the Cys-82 and Cys-153 of the β subunit were individually mutated, giving two kinds of mutants. The results show that Cys-153 maybe the active site for β subunit binding to phycocyanobilins, which is catalyzed by CpcT in A. platensis FACHB314.     

Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment.

Mutants of the cyanobacterium Synechococcus sp. PCC 7002 constructed by the insertional inactivation of either the cpcE or cpcF gene produce low levels of spectroscopically detectable phycocyanin. The majority of the phycocyanin produced in these strains appears to lack the alpha subunit phycocyanobilin (PCB) chromophore (Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R., and Bryant, D. A. (1992) J. Biol. Chem. 267, 16138-16145). Purification of the phycocyanin produced in the mutants revealed two fractions each with an aberrant absorption spectrum. Tryptic peptide maps of the major fraction showed that the alpha-84 PCB peptide was absent. The two PCB peptides derived from the beta subunit were normal. Tryptic digests of the less abundant phycocyanin fraction contained a family of bilin peptides derived from the alpha subunit. Several distinct bilin adducts were present. A major component was a mesobiliverdin adduct, a previously described product of the in vitro reaction of PCB and apophycocyanin. The same results were obtained with both the cpcE mutant and the cpcF mutant. In vitro reactions with PCB and the fractions containing apo alpha subunit showed that the alpha-84 bilin attachment site was unmodified and competent for adduct formation. Pseudo-revertants of both strains were observed to arise at high frequency. Analysis of the phycocyanin from a cpcE pseudo-revertant, which produced a near wild-type level of phycocyanin with alpha subunit carrying PCB, revealed a single amino acid substitution, alpha-Tyr129----Cys. This residue, which is conserved in all phycocyanins sequenced to date, forms part of the alpha-84 bilin binding site and lies within 5 A of alpha-Cys84. A mutated cpcA gene containing this substitution was constructed by site-directed mutagenesis and transformed, along with cpcB, into a cpcBAC deletion strain containing an insertionally inactivated cpcE. This strain produces high levels of phycocyanin and the majority of the alpha subunit carries PCB at alpha-Cys84.     

Phytochrome chromophore biosynthesis. Treatment of tetrapyrrole-deficient Avena explants with natural and non-natural bilatrienes leads to formation of spectrally active holoproteins

Etiolated Avena seedlings grown in the presence of 4-amino-5-hexynoic acid, an inhibitor of 5-aminolevulinic acid synthesis in plants, contain less than 10% of the spectrally detectable levels of phytochrome found in untreated seedlings (Elich, T.D., and Lagarias, J.C. (1988) Plant Physiol. 88, 747-751). In this study, incubation of explants from such seedlings with [14C]biliverdin IX alpha led to rapid covalent incorporation of radiolabel into a single 124-kDa polypeptide in soluble protein extracts. Immunoprecipitation experiments confirmed that this protein was phytochrome. Parallel experiments were performed with four unlabeled linear tetrapyrroles, the naturally occurring biliverdin IX alpha isomer, two non-natural isomers, biliverdin XIII alpha and biliverdin III alpha, and phycocyanobilin-the cleaved prosthetic group of the light-harvesting antenna protein C-phycocyanin. In all cases, except for the III alpha isomer of biliverdin, a time-dependent recovery of photoreversible phytochrome was observed. The newly formed phytochrome obtained after incubation with biliverdin IX alpha exhibited spectral characteristics identical with those of the native protein. In contrast, the spectral properties of phytochromes formed during incubation with biliverdin XIII alpha and phycocyanobilin differed significantly from those of the native chromoprotein. These results indicate that biliverdin IX alpha is an intermediate in the biosynthesis of the phytochrome chromophore and that phytochromes with prosthetic groups derived from bilatrienes having non-natural D-ring substituents are photochromic.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Enzymatic heme oxygenase activity in soluble extracts of the unicellular red alga, Cyanidium caldarium

Extracts of the phycocyanin-containing unicellular red alga, Cyanidium caldarium, catalyzed enzymatic cleavage of the heme macrocycle to form the linear tetrapyrrole bilin structure. This is the key first step in the branch of the tetrapyrrole biosynthetic pathway leading to phycobilin photosynthetic accessory pigments. A mixed-function oxidase mechanism, similar to the biliverdin-forming reaction catalyzed by animal cell-derived microsomal heme oxygenase, was indicated by requirements for O2 and a reduced pyridine nucleotide. To avoid enzymatic conversion of the bilin product to phycocyanobilins and subsequent degradation during incubation, mesoheme IX was substituted for the normal physiological substrate, protoheme IX. Mesobiliverdin IX alpha was identified as the primary incubation product by comparative reverse-phase high-pressure liquid chromatography and absorption spectrophotometry. The enzymatic nature of the reaction was indicated by the requirement for cell extract, absence of activity in boiled cell extract, high specificity for NADPH as cosubstrate, formation of the physiologically relevant IX alpha bilin isomer, and over 75% inhibition by 1 microM Sn-protoporphyrin, which has been reported to be a competitive inhibitor of animal microsomal heme oxygenase. On the other hand, coupled oxidation of mesoheme, catalyzed by ascorbate plus pyridine or myoglobin, yielded a mixture of ring-opening mesobiliverdin IX isomers, was not inhibited by Sn-protoporphyrin, and could not use NADPH as the reductant. Unlike the animal microsomal heme oxygenase, the algal reaction appeared to be catalyzed by a soluble enzyme that was not sedimentable by centrifugation for 1 h at 200,000g. Although NADPH was the preferred reductant, small amounts of activity were obtained with NADH or ascorbate. A portion of the activity was retained after gel filtration of the cell extract to remove low-molecular-weight components. Considerable stimulation of activity, particularly in preparations that had been subjected to gel filtration, was obtained by addition of ascorbate to the incubation mixture containing NADPH. The results indicate that C. caldarium possesses a true heme oxygenase system, with properties somewhat different from that catalyzing heme degradation in animals. Taken together with previous results indicating that biliverdin is a precursor to phycocyanobilin, the results suggest that algal heme oxygenase is a component of the phycobilin biosynthetic pathway.     

Integration of Apo-α-Phycocyanin into Phycobilisomes and Its Association with FNRL in the Absence of the Phycocyanin α-Subunit Lyase (CpcF) in Synechocystis sp. PCC 6803

Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR_L) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR_L accumulated. FNR_L has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNR_L and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.