A cyanobacterial phytochrome two-component light sensory system.

The biliprotein phytochrome regulates plant growth and developmental responses to the ambient light environment through an unknown mechanism. Biochemical analyses demonstrate that phytochrome is an ancient molecule that evolved from a more compact light sensor in cyanobacteria. The cyanobacterial phytochrome Cph1 is a light-regulated histidine kinase that mediates red, far-red reversible phosphorylation of a small response regulator, Rcp1 (response regulator for cyanobacterial phytochrome), encoded by the adjacent gene, thus implicating protein phosphorylation-dephosphorylation in the initial step of light signal transduction by phytochrome.

PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis.

Plants constantly monitor their light environment in order to grow and develop optimally, in part through use of the phytochromes, which sense red/far-red light. A phytochrome binding protein, PKS1 (phytochrome kinase substrate 1), was identified that is a substrate for light-regulated phytochrome kinase activity in vitro. In vivo experiments suggest that PKS1 is phosphorylated in a phytochrome-dependent manner and negatively regulates phytochrome signaling. The data suggest that phytochromes signal by serine-threonine phosphorylation.

The phytofluors: a new class of fluorescent protein probes.

BACKGROUND: Biologically compatible fluorescent protein probes, particularly the self-assembling green fluorescent protein (GFP) from the jellyfish Aequorea victoria, have revolutionized research in cell, molecular and developmental biology because they allow visualization of biochemical events in living cells. Additional fluorescent proteins that could be reconstituted in vivo while extending the useful wavelength range towards the orange and red regions of the light spectrum would increase the range of applications currently available with fluorescent protein probes. RESULTS: Intensely orange fluorescent adducts, which we designate phytofluors, are spontaneously formed upon incubation of recombinant plant phytochrome apoproteins with phycoerythrobilin, the linear tetrapyrrole precursor of the phycoerythrin chromophore. Phytofluors have large molar absorption coefficients, fluorescence quantum yields greater than 0.7, excellent photostability, stability over a wide range of pH, and can be reconstituted in living plant cells. CONCLUSIONS: The phytofluors constitute a new class of fluorophore that can potentially be produced upon bilin uptake by any living cell expressing an apophytochrome cDNA. Mutagenesis of the phytochrome apoprotein and/or alteration of the linear tetrapyrrole precursor by chemical synthesis are expected to afford new phytofluors with fluorescence excitation and emission spectra spanning the visible to near-infrared light spectrum.

The structure of verdohemochrome and its implications for the mechanism of heme catabolism.

The synthesis, purification as a tetrafluoroborate salt and structural elucidation of the verdohemochrome 2a derived from the coupled oxidation of octaethylhemochrome 1 is described. Based on elemental analyses, spectroscopic studies (visible and infrared absorption, 1H-NMR) and fast atom bombardment mass spectrometry, the assignment of the iron(II) oxaporphyrin structure for the verdohemochrome 2a and the blue monocarbonyl species 2b, obtained upon treatment of 2a with carbon monoxide, has been accomplished. This assignment raises a number of questions regarding the iron oxidation state of intermediates in the pathway of heme catabolism both in vitro and in vivo. Furthermore, the implications of the occurrence of an iron oxaporphyrin intermediate in the pathway of heme metabolism, which is suggested by the similarity of the visible absorption spectrum of the CO species 2b with that of a new intermediate recently observed in the heme oxygenase-catalyzed degradation of heme and mesoheme, is considered.

Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A.

Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity. Expression of an ASPHYA cDNA that encoded the full-length photoreceptor with a 15 amino acid 'strep-tag' peptide at its C-terminus produced a single polypeptide with a molecular mass of 124 kDa. This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45%. Incubation of ASPHYA-ST with phytochromobilin (P phi B) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified. Both P phi B and PCB adducts of ASPHYA-ST were photoactive--the P phi B adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor, and the PCB adduct exhibiting blue-shifted absorption maxima. Although the PEB adduct of ASPHYA-ST was photochemically inactive, it was intensely fluorescent with an excitation maximum at 576 nm and emission maxima at 586 nm. The superimposability of its absorption and fluorescence excitation spectra established that a single biliprotein species was responsible for fluorescence from the adduct produced when ASPHYA-ST was incubated with PEB. Steric exclusion HPLC also confirmed that ASPHYA-ST and its three bilin adducts were homodimers, as has been established for phytochrome A isolated from natural sources. The ability to express and purify recombinant phytochromes with biochemical properties very similar to those of the native molecule should facilitate detailed structural analysis of this important class of photoreceptors.

Bile pigment--protein interactions. Coupled oxidation of cytochrome c.

A new methodology is described for the chemical modification of the heme prosthetic group of horse heart cytochrome c. The selective modification of the heme moiety of cytochrome c is facilitated by utilizing coupling oxidation conditions. Comparison of the absorption spectra of this chemically modified cytochrome c species in two different solvents (aqueous pyridine and carbon monoxide saturated 6 M guanidinium chloride) with those of two model compounds [bis(pyridine)(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt and (pyridine)carbonyl-(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt] shows that coupled oxidation of cytochrome c affords a new protein with a covalently bound iron(II) oxaporphyrin prosthetic group. Amino acid analysis of this protein-bound iron(II) oxaporphyrin species reveals that only limited modification of the primary structure of the apoprotein occurs during coupled oxidation of cytochrome c. This protein-bound iron(II) oxaporphyrin species is also interconvertible to a protein-bound bilatriene species under hydrolytic conditions. The synthetic utility of the coupled oxidation of cytochrome c for the preparation of chromoproteins which possess covalently bound iron(II) oxaporphyrin and bilatriene prosthetic groups is considered.

Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro.

Light-mediated conformational changes in highly purified 124-kDa phytochrome preparations from etiolated oat seedlings have been identified by steric exclusion high performance liquid chromatography and limited proteolytic studies. Steric exclusion high performance liquid chromatography studies of oat and rye phytochromes show photoreversible changes in retention times, with the red absorbing form of phytochrome (Pr form) eluting later than the far red absorbing form of phytochrome produced by saturating red light illumination of Pr (Pfr form) in a variety of different mobile phase buffers. Molecular mass calibration with globular protein standards in Tris-glycol buffers provides estimates of 318-349 and 363-366 kDa for the molecular sizes of the Pr and Pfr forms, respectively. These analyses support earlier studies that phytochrome is a nonglobular homodimer of 124-kDa subunits in vitro. Limited proteolytic dissection of phytochrome in nondenaturing buffers with seven different endoproteases provides evidence for two "operational" domains within the 124-kDa subunit with molecular mass values of 69-72 and 52-55 kDa. The larger 69-72-kDa domain contains the site for the chromophore attachment as shown by gel electrophoresis derived enzyme-linked immunosorbent assay utilizing site-directed rabbit antiserum to a synthetic undecapeptide which is homologous with the chromophore binding site on oat phytochrome. This chromophore domain exhibits a compact structure, resistant to further proteolysis except near its N terminus. By contrast, the 52-55-kDa nonchromophore domain contains multiple sites for further proteolytic cleavage as revealed by rapid cleavage to smaller polypeptide fragments. Detailed kinetic analyses of the limited proteolytic cleavage of phytochrome with four endoproteases, subtilisin BPN', thermolysin, trypsin, and clostripain, has mapped specific regions within the 124-kDa subunit that participate in light-induced conformational changes. These include a 4-10-kDa region near the N terminus of the chromophore binding domain and at least two regions within the nonchromophore domain. A comprehensive peptide map of the oat phytochrome subunit is presented, which incorporates the results of these proteolytic studies with the recent, yet unpublished sequence analyses of Avena phytochrome cDNA clones which show the N-terminal localization of the chromophore binding site (Hershey, H. P., Colbert, J. T., Lissemore, J. L., Barker, R. F., and Quail, P. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2332-2336).

4-amino-5-hexynoic Acid-a potent inhibitor of tetrapyrrole biosynthesis in plants.

4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants.

Phytochrome assembly. Defining chromophore structural requirements for covalent attachment and photoreversibility.

Assembly of holophytochrome in the plant cell requires covalent attachment of the linear tetrapyrrole chromophore precursor, phytochromobilin, to a unique cysteine in the nascent apoprotein. In this investigation we compare chromophore analogs with the natural chromophore precursor for their ability to attach covalently to recombinant oat apophytochrome and to form photoactive holoproteins. Ethylidene-containing analogs readily form covalent adducts with apophytochrome, whereas chromophores lacking this double bond are poor substrates for attachment. Kinetic measurements establish that although the chromophore binding site on apophytochrome is best tailored to phytochromobilin, apophytochrome will accommodate the two analogs with modified D-rings, phycocyanobilin and phycoerythrobilin. The phycocyanobilin-apophytochrome adduct is photoactive and undergoes a light-induced protein conformational change similar to the native holoprotein. By contrast, the phycoerythrobilin adduct is locked into a photochemically inactive protein conformation that is similar to the red light-absorbing Pr form of phytochrome. These results support the hypothesis that the photoconversion from Pr to Pfr, the far red light- absorbing form of phytochrome, involves the photoisomerization of the C15 double bond. Knowledge gained from these studies provides impetus for rational design of chromophore analogs whose insertion into apophytochrome should elicit profound changes in light-mediated plant growth and development.

Defining the bilin lyase domain: lessons from the extended phytochrome superfamily.

Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified and used to construct a 130-180 amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins). To test this hypothesis, portions of locus sll0821, a novel phytochrome-related gene from Synechocystis sp. PCC6803 that encodes a large protein with two potential bilin binding sites, were amplified, and the recombinant apoproteins were tested for bilin binding and phytochrome photoactivity. Our experiments indicated that both sites of this protein, termed Cph2 for cyanobacterial phytochrome 2, possessed bilin lyase activity, revealing two distinct classes of bilin lyase domains--those whose bilin adducts are red, far-red reversible and a second class whose bilin adducts are nonphotochromic. Spectroscopic analysis of photochromic phycocyanobilin and fluorescent phycoerythrobilin adducts of a 24-kDa fragment of Cph2 definitively established that the motif identified by pattern searches represents a bona fide bilin lyase domain. Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding.

Genetic engineering of phytochrome biosynthesis in bacteria.

The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.

Purification and biochemical properties of phytochromobilin synthase from etiolated oat seedlings.

Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (P Phi B) for photoactivity. In planta, biliverdin IX alpha (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme P Phi B synthase to yield 3Z-P Phi B. Here, we describe the >50,000-fold purification of P Phi B synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, P Phi B synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s(-1), which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat P Phi B synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of P Phi B synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A K(m) for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 microM. P Phi B synthase has a high affinity for its bilin substrate, with a sub-micromolar K(m) for BV.

Phytochrome from the green alga Mesotaenium caldariorum. Purification and preliminary characterization.

A method to purify the phytochrome photoreceptor from the unicellular green alga Mesotaenium caldariorum is presented. Preparative scale formation of algal protoplasts and controlled osmotic cell lysis have permitted separation of intact organelles from the phytochrome-enriched soluble protein fraction. We have utilized the observation that red light-absorbing (Pr) and far-red light-absorbing (Pfr) forms of phytochrome are differentially retained on an anion exchange matrix to purify M. caldariorum phytochrome to apparent homogeneity. M. caldariorum phytochrome preparations with A650/A280 ratios greater than 0.78 exhibit a single 120-kDa band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses using a cross-reactive pea phytochrome monoclonal antibody reveal that 1) the 120-kDa band represents the full-length polypeptide, 2) phytochrome is predominantly localized in the algal cytoplasm, and 3) there are 150,000-250,000 phytochrome molecules/cell. Steric exclusion high pressure liquid chromatography analysis under nondenaturing conditions indicates that M. caldariorum phytochrome has an apparent mass of 355 kDa. The absorption maxima for Pr and Pfr are 650 and 722 nm, respectively. Both are blue-shifted compared with those of phytochromes from dark-grown angiosperm tissue. The molar absorption coefficient for Pr at 650 nm is 86,800 +/- 2800 liter mol-1 cm-1, which is lower than that of higher plant phytochromes. The significance of the similarities and differences of the molecular properties of phytochromes from M. caldariorum and higher plant sources is discussed.

Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution.

The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca(2+) per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I(50) values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I(50) of 25 micromolar). These results suggest that direct Ca(2+) transport and Ca(2+)/H(+) antiport activities are present in both sucrose gradient fractions.

Holophytochrome assembly. Coupled assay for phytochromobilin synthase in organello.

Utilizing an in vitro coupled assay system, we show that isolated plastids from cucumber cotyledons convert the linear tetrapyrrole biliverdin IX alpha to the free phytochrome chromophore, phytochromobilin, which assembles with oat apophytochrome to yield photoactive holoprotein. The spectral properties of this synthetic phytochrome are indistinguishable from those of the natural photoreceptor. The plastid-dependent biliverdin conversion activity is strongly stimulated by both NADPH and ATP. Substitution of the nonnatural XIII alpha isomer of biliverdin for the IX alpha isomer affords a synthetic holophytochrome adduct with blue-shifted difference spectra. These results, together with experiments using boiled plastids, indicate that phytochromobilin synthesis from biliverdin is enzyme-mediated. Experiments where NADPH (and ATP) levels in intact developing chloroplasts are manipulated by feeding the metabolites 3-phosphoglycerate, dihydroxyacetone phosphate, and glucose 6-phosphate or by illumination with white light, support the hypothesis that the enzyme that accomplishes this conversion, phytochromobilin synthase, is plastid-localized. It is therefore likely that all of the enzymes of the phytochrome chromophore biosynthetic pathway reside in the plastid.

Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry.

The discovery of cyanobacterial phytochrome histidine kinases, together with the evidence that phytochromes from higher plants display protein kinase activity, bind ATP analogs, and possess C-terminal domains similar to bacterial histidine kinases, has fueled the controversial hypothesis that the eukaryotic phytochrome family of photoreceptors are light-regulated enzymes. Here we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings. Phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process. Based on comparative protein sequence alignments and biochemical cross-talk experiments with the response regulator substrate of the cyanobacterial phytochrome Cph1, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module.

Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae.

The biological activity of the plant photoreceptor phytochrome requires the specific association of a linear tetrapyrrole prosthetic group with a large apoprotein. As an initial step to develop an in vivo assay system for structure-function analysis of the phytochrome photoreceptor, we undertook experiments to reconstitute holophytochrome in the yeast Saccharomyces cerevisiae. Here we show that yeast cells expressing recombinant oat apophytochrome A can take up exogenous linear tetrapyrroles, and, in a time-dependent manner, these pigments combine with the apoprotein to form photoactive holophytochrome in situ. Cell viability measurements indicate that holophytochrome assembly occurs in living cells. Unlike phytochrome A in higher plant tissue, which is rapidly degraded upon photoactivation, the reconstituted photoreceptor appears to be light stable in yeast. Reconstitution of photoactive phytochrome in yeast cells should enable us to exploit the power of yeast genetics for structure-function dissection of this important plant photoreceptor.

Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro.

Avena seedlings grown in the presence of the plant tetrapyrrole synthesis inhibitor 4-amino-5-hexynoic acid contain less than 10% of the spectrally detectable phytochrome levels found in untreated seedlings, but continue to accumulate phytochrome apoprotein (Elich, T. D., and Lagarias, J. C. (1988) Plant Physiol. 88, 747-751). Using such tetrapyrrole-deficient seedlings, we have previously reported that phycocyanobilin, the cleaved prosthetic group of C-phycocyanin, can be incorporated into phytochrome in vivo to yield spectrally active holoprotein (Elich, T. D., McDonagh, A. F., Palma, L. A., and Lagarias, J. C. (1988) J. Biol. Chem. 264, 183-189). Here we show that addition of phycocyanobilin to soluble extracts of inhibitor-treated seedlings results in a rapid increase in spectrally active phytochrome holoprotein. The newly formed photoactive species displays a blue-shifted absorbance difference spectrum similar to that observed in the previous in vivo studies. The increase in spectral activity is consistent with conversion of all of the preexisting phytochrome apoprotein to functionally active holoprotein. The formation of a covalent phycocyanobilin-apophytochrome adduct is shown by an increase in Zn2+-dependent bilin fluorescence of the phytochrome polypeptide. A photoreversible, covalent adduct with a similar optical spectrum also forms when immunopurified apophytochrome is incubated with phycocyanobilin. ATP, reduced pyridine nucleotides, or other cofactors are not required for adduct formation. When biliverdin IX alpha is substituted for phycocyanobilin, no spectrally active covalent adduct is produced. These results indicate that an A-ring ethylidene-containing bilatriene is required for post-translational covalent attachment of bilin to apophytochrome and that apophytochrome may be the bilin C-S lyase which catalyzes bilin attachment.

Affinity labeling of Avena phytochrome with ATP analogs.

The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5'-triphosphate, were used to affinity label purified Avena phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys(75),Ala(25)) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome.