The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

Revisiting high-resolution crystal structure of Phormidium rubidum phycocyanin.

The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in ß-subunit of pr-PC), in which all residues except one (ß145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified pr-PC was then sequenced by mass spectrometry (MS) using LC-MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of pr-PC allows the chromophore interactions with the protein to be described with high accuracy.

Spectroscopic and molecular docking studies on the interaction of phycocyanobilin with peptide moieties of C-phycocyanin.

The binding of C-phycocyanin (CPC), a light harvesting pigment with phycocyanobilin (PCB), a chromophore is instrumental for the coloration and bioactivity. In this study, structure-mediated color changes of CPC from Spirulina platensis during various enzymatic hydrolysis was investigated based on UV-visible, circular dichroism, infra-red, fluorescence, mass spectrometry, and molecular docking. CPC was hydrolyzed using 7.09 U/mg protein of each enzyme at their optimal hydrolytic conditions for 3 h as follows: papain (pH 6.6, 60 °C), dispase (pH 6.6, 50 °C), and trypsin (pH 7.8, 37 °C). The degree of hydrolysis was in the order of papain (28.4%) > dispase (20.8%) > trypsin (7.3%). The sequence of color degradation rate and total color difference (ΔE) are dispase (82.9% and 40.37), papain (72.4% and 24.70), and trypsin (58.7% and 25.43). The hydrolyzed peptides were of diverse sequence length ranging from 8 to 9 residues (papain), 7-12 residues (dispase), and 9-63 residues (trypsin). Molecular docking studies showed that key amino acid residues in the peptides interacting with chromophore. Amino acid residues such as Arg86, Asp87, Tyr97, Asp152, Phe164, Ala167, and Val171 are crucial in hydrogen bonding interaction. These results indicate that the color properties of CPC might associate with chromopeptide sequences and their non-covalent interactions.

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore.

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical "transparency window" of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.

Antioxidant activity of proteins extracted from red alga dulse harvested in Japan.

In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.

Phycobiliproteins from cyanobacteria: Chemistry and biotechnological applications.

Phycobiliproteins are a group of water soluble proteins with an associated chromophore, responsible for the light-harvesting in cyanobacteria. They are divided in four main types: phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin, and they are characterized according to their structure and light quality absorption. Phycobiliproteins from cyanobacteria have been described as potential bioactive compounds, and recognized as high-valued natural products for biotechnological applications. Moreover, phycobiliproteins have been associated to antioxidant, anticancer and anti-inflammatory capacities among others. Thus, in order to produce phycobiliproteins from cyanobacteria for industrial application, it is necessary to optimize the whole bioprocess, including the processing parameters (such as light, nitrogen and carbon source, pH, temperature and salinity) that affects the growth and phycobiliprotein accumulation, as well as the optimization of phycobiliproteins extraction and purification. The aim of this review is to give an overview of phycobiliproteins not only in terms of their chemistry, but also in terms of their biotechnological applicability and the advances and challenges in the production of such compounds.

Determination of the protonation preferences of bilin pigments in cryptophyte antenna complexes.

The light-harvesting mechanisms of cryptophyte antenna complexes have attracted considerable attention due to their ability to exhibit maximal photosynthetic activity under very low-light conditions and to display several colors, as well as the observation of vibronic coherent features in their two-dimensional electronic spectra. However, detailed investigations on the interplay between the protein environment and their light-harvesting properties are hampered by the uncertainty related to the protonation state of the underlying bilin pigments. Here we study the protonation preferences of four types of bilin pigments including 15,16-dihydrobiliverdin (DBV), phycoerythrobilin (PEB), phycocyanobilin (PCB) and mesobiliverdin (MBV), which are found in phycoerythrin PE545 and phycocyanin PC577, PC612, PC630 and PC645 complexes. We apply quantum chemical calculations coupled to continuum solvation calculations to predict the intrinsic acidity of bilins in aqueous solution, and then combine molecular dynamics simulations with empirical pKa estimates to investigate the impact of the local protein environment on the acidity of the pigments. We also report measurements of the absorption spectra of the five complexes in a wide range of pH in order to validate our simulations and investigate possible changes in the light harvesting properties of the complexes in the range of physiological pH found in the lumen (pH ∼ 5-7). The results suggest a pKa > 7 for DBV and MBV pigments in the α polypeptide chains of PE545 and PC630/PC645 complexes, which are not coordinated to a negatively charged amino acid. For the other PEB, DBV and PCB pigments, which interact with a Glu or Asp side chain, higher pKa values (pKa > 8) are estimated. Overall, the results support a preferential population of the fully protonated state for bilins in cryptophyte complexes under physiological conditions regardless of the specific type of pigment and local protein environment.

Covalent binding of food-derived blue pigment phycocyanobilin to bovine ß-lactoglobulin under physiological conditions.

In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine ß-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (ka = 0.065 min-1), of moderate affinity (Ka = 4 × 104 M-1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (ka = 0.101 min-1). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB.

Beneficial effects of oral administration of C-Phycocyanin and Phycocyanobilin in rodent models of experimental autoimmune encephalomyelitis.

The only three oral treatments currently available for multiple sclerosis (MS) target the relapsing forms of the disease and concerns regarding efficacy, safety and tolerability limit their use. Identifying novel oral disease-modifying therapies for MS, targeting both its inflammatory and neurodegenerative components is still a major goal. AIM: The scope of this study was to provide evidence that the oral administration of C-Phycocyanin (C-PC), the main biliprotein of the Spirulina platensis cyanobacteria and its tetrapyrrolic prosthetic group, Phycocyanobilin (PCB), exert ameliorating actions on rodent models of experimental autoimmune encephalomyelitis (EAE). MAIN METHODS: EAE was induced in Lewis rats using the spinal cord encephalitogen from Sprague Dawley rats and in C57BL6 mice with MOG35-55 peptide. Clinical signs, motor function, oxidative stress markers, cytokine levels by ELISA and transmission electron microscopy analysis were assessed. KEY FINDINGS: Either prophylactic or early therapeutic administration of C-PC to Lewis rats with EAE, significantly improved clinical signs and restored the motor function of the animals. Furthermore, C-PC positively modulated oxidative stress markers measured in brain homogenate and serum and protected the integrity of cerebral myelin sheaths as shown by transmission electron microscopy analysis. In C57BL/6 mice with EAE, PCB orally improved clinical status of the animals and reduced the expression levels of brain IL-6 and IFN-γ proinflammatory cytokines. SIGNIFICANCE: These results, for the first time, support the fact that both C-PC and PCB administered orally could potentially improve neuroinflammation, protect from demyelination and axonal loss, which may be translated into an improved quality of life for MS patients.

Phycobilins and Phycobiliproteins Used in Food Industry and Medicine.

BACKGROUND: Open tetrapyrroles termed phycobilins represent the major photosynthetic accessory pigments of several cyanobacteria and some eukaryotic algae such as the Glaucophyta, Cryptophyta and Rhodophyta. These pigments are covalently bound to so-called phycobiliproteins which are in general organized into phycobilisomes on the thylakoid membranes. OBJECTIVE & METHODS: In this work we first briefly describe the physico-chemical properties, biosynthesis, occurrence, in vivo localization and roles of the phycobilin pigments and the phycobiliproteins. Then the potential applications and uses of these pigments, pigment-protein complexes and related products by the food industry (e.g., as LinaBlue® or the so-called spirulina extract used as coloring food), by the health industry or as fluorescent dyes are critically reviewed. CONCLUSION: In addition to the stability, bioavailability and safety issues of purified phycobilins and phycobiliproteins, literature data about their antioxidant, anticancer, anti-inflammatory, immunomodulatory, hepatoprotective, nephroprotective and neuroprotective effects, and their potential use in photodynamic therapy (PDT) are also discussed.

Carvedilol and spirulina may provide important health protection to smokers and other nicotine addicts: a call for pertinent research.

Nicotine and soluble semi-stable aldehydes and ketones in cigarette smoke are key mediators of the elevated risks for vascular disease, cancer, and chronic obstructive pulmonary disease observed in smokers. Nicotine, via sympathetic stimulation, increases risk for both vascular disease and cancer. Comprehensive suppression of sympathetic activity with the well-tolerated drug carvedilol, which inhibits betal 1, beta2 and alphal adrenergic receptors, may be protective to smokers and other nicotine addicts. The soluble aldehydes and ketones in tobacco smoke appear to exert their adverse effects through activation of NADPH oxidase complexes in vascular tissues and in the lungs. The phytochemical phycocyanobilin (PhyCB), richly supplied by the edible cyanobacterium spirulina, in studies on rodents and in human cell cultures has shown the ability to safely mimic intracellular bilirubin's physiological role as an inhibitor ofNADPH oxidase activity. It therefore may have potential for mitigating the pro-oxidative effects of tobacco smoke aldehydes and ketones. Joint administration of carvedilol and spirulina merits exploration as a strategy for moderating the pathogenic impact of smoking in chronic tobacco users who either fail to quit or refuse to try cessation of tobacco. Carvedilol may be appropriate for those who manage a nicotine addiction in other ways (smokeless tobacco, e-cigarettes, nicotine gum). Further clinical studies to evaluate the impact of carvedilol on cardiovascular risk factors in nicotine addicts, and rodent studies to assess markers of lung inflammation in smoke- exposed rodents fed PhyCB, are recommended.

Comprehensive analysis of the green-to-blue photoconversion of full-length Cyanobacteriochrome Tlr0924.

Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 ?s. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms.

Direct differentiation of A-ring single attachment versus A- and D-ring double attachment of phycoerythrobilin chromophores to phycobiliproteins using MALDI mass spectrometry.

Bilin chromophore attachment to phycobiliproteins is an enzyme-catalyzed post-translational modification process. Bilin-lyases attach a bilin chromophore to their cognate protein through a thioether bond between the chromophore and a cysteine moiety. Bilin chromophores are attached to their phycobiliproteins through the 3(1) carbon of the bilin. Double attachment may also occur, and in this case, carbons 3(1) and 18(1) of the bilin are both forming covalent linkages to cysteine moieties. There is a mass spectrometric limitation when examining tryptic peptides containing two (or more) cysteines if one seeks to ascertain whether chromopeptides are singly or doubly attached. The problem is that singly and doubly attached chromopeptides appear at the same m/z value; thus, up until the present, only NMR analysis has been successful at determining whether the chromophore is singly or doubly attached. We report in this work a new, fast and accurate method for discriminating singly from doubly attached chromophores using MALDI-TOF mass spectrometry. This method was developed from mass spectral analysis of chromopeptides that had undergone in vitro or in vivo attachment of bilin chromophores to phycobiliproteins. Distinction is based on a characteristic neutral loss that appears in the MALDI-TOF mass spectrum only when the bilin is singly attached.

Phycoviolobilin formation and spectral tuning in the DXCF cyanobacteriochrome subfamily.

Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.

Marinobufagenin and cyclic strain may activate endothelial NADPH oxidase, contributing to the adverse impact of salty diets on vascular and cerebral health.

Limited but provocative ecologic epidemiology suggests that dietary salt may play a central role in the genesis of not only of stroke, but also dementia, including Alzheimer's disease. Impairment of nitric oxide bioactivity in the cerebral microvasculature is a likely mediator of this effect. Salted diets evoke increased adrenal secretion of the natriuretic steroid marinobufagenin (MBG), which promotes natriuresis via inhibition of renal tubular Na+/K+-ATPase; this effect is notably robust in salt-sensitive rodent strains in which other compensatory natriuretic mechanisms are subnormally efficient. MBG-mediated inhibition of sodium pumps in vascular smooth muscle likely plays a role in the hypertension induced by salty diets in these rodents. However, salt sensitivity in humans is associated with increased vascular mortality and ventricular hypertrophy independent of blood pressure; this suggests that MBG may be pathogenic via mechanisms unrelated to blood pressure control. Indeed, recent evidence indicates that MBG, via interaction with alpha1 isoforms of the sodium pump, can activate various intracellular signaling pathways at physiological concentrations too low to notably inhibit pump activity. An overview of current evidence suggests the hypothesis that MBG - as well as the cyclic strain induced by hypertension per se - may induce endothelial oxidative stress by activating NADPH oxidase. If so, this could rationalize the increase in vascular and systemic oxidative stress observed in salt-sensitive rodents fed salty diets, or in rodents infused with MBG; moreover, if this effect is a particularly prominent determinant of oxidative stress in cerebrovascular endothelium, it might help to explain the virtual absence of stroke and dementia in low-salt societies. As a corollary of this hypothesis, it can be predicted that spirulina-derived phycobilins, which appear to mimic the physiological role of bilirubin as an inhibitor of NAPDH oxidase complexes, may have potential for ameliorating the adverse health impacts of MBG and of salty diets. Potassium-rich diets are also likely to be protective in this regard, as they should suppress MBG production via their natriuretic impact, while their stimulatory effect on sodium pump activity may exert a hyperpolarizing effect on plasma membranes that suppresses NADPH oxidase activity.

Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores: photochemistry and dark reversion kinetics.

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (λ(max) = 650 and 535 nm) that is assigned to the reversible 15Z/E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (λ(max) = 685 nm) reverting slowly thermally to the thermostable 15Z state (λ(max) = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, λ(max) = 595 nm) reverts very rapidly (τ ~ 20 s) back to the thermostable Z state (λ(max) = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1/E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1/Z-GAF3 has intermediate activity, and Z-GAF1/Z-GAF3 is the least active state.

Catalytic mechanism of S-type phycobiliprotein lyase: chaperone-like action and functional amino acid residues.

The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Plöscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300-14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a non-competitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-beta84-bound PCB in biliproteins by PEB.

Cyanochromes are blue/green light photoreversible photoreceptors defined by a stable double cysteine linkage to a phycoviolobilin-type chromophore.

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3(1) carbon of the ethylidene side chain and the C4 or C5 carbons in the A-B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb --> Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.

Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyase-isomerase.

Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (lambda(max) approximately 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its alpha-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters.