Biomedical subjects

J C Lagarias

Publications and source records attributed to J C Lagarias.

At least 19 recordsLinked to original sources

PubMed2001

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.

Apoproteins↗

PubMed2001

Biliverdin reductase-induced phytochrome chromophore deficiency in transgenic tobacco.

Targeted expression of mammalian biliverdin IXalpha reductase (BVR), an enzyme that metabolically inactivates linear tetrapyrrole precursors of the phytochrome chromophore, was used to examine the physiological functions of phytochromes in the qualitative short-day tobacco (Nicotiana tabacum cv Maryland Mammoth) plant. Comparative phenotypic and photobiological analyses of plastid- and cytosol-targeted BVR lines showed that multiple phytochrome-regulated processes, such as hypocotyl and internode elongation, anthocyanin synthesis, and photoperiodic regulation of flowering, were altered in all lines examined. The phytochrome-mediated processes of carotenoid and chlorophyll accumulation were strongly impaired in plastid-targeted lines, but were relatively unaffected in cytosol-targeted lines. Under certain growth conditions, plastid-targeted BVR expression was found to nearly abolish the qualitative inhibition of flowering by long-day photoperiods. The distinct phenotypes of the plastid-targeted BVR lines implicate a regulatory role for bilins in plastid development or, alternatively, reflect the consequence of altered tetrapyrrole metabolism in plastids due to bilin depletion.

Agrobacterium tumefaciens↗

PubMed2001

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.

Avena↗

PubMed2001

The Arabidopsis HY2 gene encodes phytochromobilin synthase, a ferredoxin-dependent biliverdin reductase.

Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (P Phi B), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2, which are defective in P Phi B biosynthesis, display altered responses to light due to a deficiency in photoactive phytochrome. Here, we describe the isolation of the HY2 gene by map-based cloning. hy2 mutant alleles possess alterations within this locus, some of which affect the expression of the HY2 transcript. HY2 encodes a soluble protein precursor of 38 kD with a putative N-terminal plastid transit peptide. The HY2 transit peptide is sufficient to localize the reporter green fluorescent protein to plastids. Purified mature recombinant HY2 protein exhibits P Phi B synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IX alpha to P Phi B), as confirmed by HPLC and by the ability of the bilin reaction products to combine with apophytochrome to yield photoactive holophytochrome. Database searches and hybridization studies suggest that HY2 is a unique gene in the Arabidopsis genome that is related to a family of proteins found in oxygenic photosynthetic bacteria.

Alleles↗

PubMed2001

Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms.

Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae, and cryptomonads. Previous biochemical studies have established that phytobilins are synthesized from heme via the intermediacy of biliverdin IX alpha (BV), which is reduced subsequently by ferredoxin-dependent bilin reductases with different double-bond specificities. By exploiting the sequence of phytochromobilin synthase (HY2) of Arabidopsis, an enzyme that catalyzes the ferredoxin-dependent conversion of BV to the phytochrome chromophore precursor phytochromobilin, genes encoding putative bilin reductases were identified in the genomes of various cyanobacteria, oxyphotobacteria, and plants. Phylogenetic analyses resolved four classes of HY2-related genes, one of which encodes red chlorophyll catabolite reductases, which are bilin reductases involved in chlorophyll catabolism in plants. To test the catalytic activities of these putative enzymes, representative HY2-related genes from each class were amplified by the polymerase chain reaction and expressed in Escherichia coli. Using a coupled apophytochrome assembly assay and HPLC analysis, we examined the ability of the recombinant proteins to catalyze the ferredoxin-dependent reduction of BV to phytobilins. These investigations defined three new classes of bilin reductases with distinct substrate/product specificities that are involved in the biosynthesis of the phycobiliprotein chromophore precursors phycoerythrobilin and phycocyanobilin. Implications of these results are discussed with regard to the pathways of phytobilin biosynthesis and their evolution.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Probing the photoreaction mechanism of phytochrome through analysis of resonance Raman vibrational spectra of recombinant analogues.

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C(15)-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-)(1) in P(r) and P(fr), respectively, are assigned as C(15)-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-)(1) in P(r) and at 672 and 654 cm(-)(1) in P(fr) are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximately 1300 cm(-)(1) in P(r) are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P(r) and P(fr) are C(15)-Z,syn and C(15)-E,anti, respectively. The intensities of the C(15)-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P(r) and P(fr) suggest that the initial photochemistry involves simultaneous bond rotations at the C(15)-methine bridge coupled to C(15)-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C(15)-E,anti P(fr) chromophore structure indicated by the intense 814 cm(-1) C(15) HOOP mode suggest that the excited state of P(fr) and its photoproduct states are strongly coupled.

Avena↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Modification of distinct aspects of photomorphogenesis via targeted expression of mammalian biliverdin reductase in transgenic Arabidopsis plants.

The phenotypic consequences of targeted expression of mammalian biliverdin IXalpha reductase (BVR), an enzyme that metabolically inactivates the linear tetrapyrrole precursors of the phytochrome chromophore, are addressed in this investigation. Through comparative phenotypic analyses of multiple plastid-targeted and cytosolic BVR transgenic Arabidopsis plant lines, we show that the subcellular localization of BVR affects distinct subsets of light-mediated and light-independent processes in plant growth and development. Regardless of its cellular localization, BVR suppresses the phytochrome-modulated responses of hypocotyl growth inhibition, sucrose-stimulated anthocyanin accumulation, and inhibition of floral initiation. By contrast, reduced protochlorophyll levels in dark-grown seedlings and fluence-rate-dependent reduction of chlorophyll occur only in transgenic plants in which BVR is targeted to plastids. Together with companion analyses of the phytochrome chromophore-deficient hy1 mutant, our results suggest a regulatory role for linear tetrapyrroles within the plastid compartment distinct from their assembly with apophytochromes in the cytosol.

Agrobacterium tumefaciens↗

PubMed1998

A phytochrome from the fern Adiantum with features of the putative photoreceptor NPH1.

In plant photomorphogenesis, it is well accepted that the perception of red/far-red and blue light is mediated by distinct photoreceptor families, i.e., the phytochromes and blue-light photoreceptors, respectively. Here we describe the discovery of a photoreceptor gene from the fern Adiantum that encodes a protein with features of both phytochrome and NPH1, the putative blue-light receptor for second-positive phototropism in seed plants. The fusion of a functional photosensory domain of phytochrome with a nearly full-length NPH1 homolog suggests that this polypeptide could mediate both red/far-red and blue-light responses in Adiantum normally ascribed to distinct photoreceptors.

Amino Acid Sequence↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1997

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.

Apoproteins↗

PubMed1997

Phycocyanobilin is the natural precursor of the phytochrome chromophore in the green alga Mesotaenium caldariorum.

Compared with phytochromes isolated from etiolated higher plant tissues and a number of lower plant species, the absorption spectrum of phytochrome isolated from the unicellular green alga Mesotaenium caldariorum is blue-shifted (Kidd, D. G., and Lagarias, J. C. (1990) J. Biol. Chem. 265, 7029-7035). The present studies were undertaken to determine whether this blue shift is due to a chromophore other than phytochromobilin or reflects a different protein environment for the phytochromobilin prosthetic group. Using reversed phase high performance liquid chromatography, we show that soluble protein extracts prepared from algal chloroplasts contain the enzyme activities for ferredoxin-dependent conversions of biliverdin IXalpha to (3Z)-phytochromobilin and (3Z)-phytochromobilin to (3Z)-phycocyanobilin. In vitro assembly of recombinant algal apophytochrome was undertaken with (3E)-phytochromobilin and (3E)-phycocyanobilin. The difference spectrum of the (3E)-phycocyanobilin adduct was indistinguishable from that of phytochrome isolated from dark-adapted algal cells, while the (3E)-phytochromobilin adduct displayed red-shifted absorption maxima relative to purified algal phytochrome. These studies indicate that phycocyanobilin is the immediate precursor of the green algal phytochrome chromophore and that phytochromobilin is an intermediate in its biosynthesis in Mesotaenium.

Biliverdine↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1997

Regulation of photomorphogenesis by expression of mammalian biliverdin reductase in transgenic Arabidopsis plants.

The photoregulatory activity of the phytochrome photoreceptor requires the synthesis and covalent attachment of the linear tetrapyrrole prosthetic group phytochromobilin. Because the mammalian enzyme biliverdin IX alpha reductase (BVR) is able to functionally inactivate phytochromobilin in vitro, this investigation was undertaken to determine whether BVR expression in transgenic plants would prevent the synthesis of functionally active phytochrome in vivo. Here, we show that plastid-targeted, constitutive expression of BVR in Arabidopsis yields plants that display aberrant photomorphogenesis throughout their life cycle. Photobiological and biochemical analyses of three transgenic BVR lines exhibiting a 25-fold range of BVR expression established that the BVR-dependent phenotypes are light dependent, pleiotropic, and consonant with the loss of multiple phytochrome activities. Chlorophyll accumulation in BVR-expressing transgenic plants was particularly sensitive to increased light fluence rates, which is consistent with an important role for phytochrome in light tolerance. Under blue light, transgenic BVR plants displayed elongated hypocotyls but retained phototropic behavior and the ability to fully deetiolate. Directed BVR expression may prove to be useful for probing the cellular and developmental basis of phytochrome-mediated responses and for selective control of individual aspects of light-mediated plant growth and development.

Animals↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1996

Resonance raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome.

Resonance Raman vibrational spectra of the Pr, lumi-R, and Pfr forms of phytochrome have been obtained using low-temperature trapping and room temperature flow techniques in conjunction with shifted-excitation Raman difference spectroscopy (SERDS). The Pr to lumi-R photoconversion exhibits a thermal barrier and is completely blocked at 30 K, indicating that thermally assisted protein relaxation is necessary for the primary photochemistry. When Pr is converted to lumi-R, new bands appear in the C = C and C = N stretching regions at 1651, 1636, 1590, and 1569 cm-1, indicating that a significant structural change of the chromophore has occurred. The photoconversion also results in an 18 cm-1 decrease in the N-H rocking band in lumi-R. Normal mode calculations correlate this frequency drop with a change in the geometry of the C15 methine bridge of the phytochromobilin chromophore. Additionally, a C = N stretching mode marker band shifts from 1576 cm-1 in Pr to 1569 cm-1 in lumi-R and to 1552 cm-1 in Pfr. Normal mode calculations show that the frequency drop of this band in the lumi-R-->Pfr interconversion is an indication of a C14-C15 syn-->anti conformational change. Moderately intense hydrogen out-of-plane modes that occur at 805 cm-1 in Pr shift to 829 and 847 cm-1 upon photoconversion to lumi-R and are replaced by a very intense mode at 814 cm-1 in Pfr. These observations indicate that the C and D rings of the chromophore in Pr and lumi-R are moderately planar but that they become highly distorted in Pfr. This information suggests that the primary photochemistry in phytochrome is a Z-->E isomerization of the C15 = C16 bond of Pr giving lumi-R. This is followed by a thermal syn-->anti C14-C15 conformational relaxation to form Pfr. A four-state model is presented to explain the chromophore structural changes in Pr, lumi-R, and Pfr that uses hydrogen bonding to the surrounding protein to stabilize the high-energy Pfr C15 = C16, C14-C15, E,anti chromophore structure. This implicates an anchor and release mechanism between the chromophore and protein that might lead to altered biological signaling in the plant.

Hydrogen Bonding↗

PubMed1996

The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome.

Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins. Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group. Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein. The hypothesis that P. pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study. Soluble extracts from P. pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A. HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin. These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.

Biliverdine↗