Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Complementation of phytochrome chromophore-deficient Arabidopsis by expression of phycocyanobilin:ferredoxin oxidoreductase.

ABSTRACT: The covalently bound phytochromobilin (PphiB) prosthetic group is required for the diverse photoregulatory activities of all members of the phytochrome family in vascular plants, whereas by contrast, green algal and cyanobacterial phytochromes use the more reduced linear tetrapyrrole pigment phycocyanobilin (PCB). To assess the functional consequence of the substitution of PphiB with PCB in plants, the phytochrome chromophore-deficient hy2 mutant of Arabidopsis was transformed with a constitutively expressed pcyA gene that encodes the cyanobacterial enzyme, PCB:ferredoxin oxidoreductase. Spectroscopic analyses of extracts from etiolated seedlings revealed that PcyA expression restored photoactive phytochrome to WT levels, albeit with blue-shifted absorption maxima, while also restoring light lability to phytochrome A. Photobiological measurements indicated that PcyA expression rescued phytochrome-mediated red high-irradiance responses, low-fluence red/far-red (FR) photoreversible responses, and very-low-fluence responses, thus confirming that PCB can functionally substitute for PphiB for these photoregulatory activities. Although PcyA expression failed to rescue phytochrome A-mediated FR high-irradiance responsivity to that of WT, our studies indicate that the FR high-irradiance response is fully functional in pcyA-expressing plants but shifted to shorter wavelengths, indicating that PCB can functionally complement this phytochrome-mediated response in vascular plants.

SUBMITTER: Kami C

PROVIDER: S-EPMC327157 | biostudies-other | 2004 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Structural insights into vinyl reduction regiospecificity of phycocyanobilin:ferredoxin oxidoreductase (PcyA).

Project description:Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.

| S-EPMC2801226 | biostudies-literature

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

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

| S-EPMC1324987 | biostudies-literature

Electrostatic interaction of phytochromobilin synthase and ferredoxin for biosynthesis of phytochrome chromophore.

Project description:In plants, phytochromobilin synthase (HY2) synthesize the open chain tetrapyrrole chromophore for light-sensing phytochromes. It catalyzes the double bond reduction of a heme-derived tetrapyrrole intermediate biliverdin IXalpha (BV) at the A-ring diene system. HY2 is a member of ferredoxin-dependent bilin reductases (FDBRs), which require ferredoxins (Fds) as the electron donors for double bond reductions. In this study, we investigated the interaction mechanism of FDBRs and Fds by using HY2 and Fd from Arabidopsis thaliana as model proteins. We found that one of the six Arabidopsis Fds, AtFd2, was the preferred electron donor for HY2. HY2 and AtFd2 formed a heterodimeric complex that was stabilized by chemical cross-linking. Surface-charged residues on HY2 and AtFd2 were important in the protein-protein interaction as well as BV reduction activity of HY2. These surface residues are close to the iron-sulfur center of Fd and the HY2 active site, implying that the interaction promotes direct electron transfer from the Fd to HY2-bound BV. In addition, the C12 propionate group of BV is important for HY2-catalyzed BV reduction. A possible role for this functional group is to mediate the electron transfer by interacting directly with AtFd2. Together, our biochemical data suggest a docking mechanism for HY2:BV and AtFd2.

| S-EPMC2836108 | biostudies-literature

Structure of the biliverdin radical intermediate in phycocyanobilin:ferredoxin oxidoreductase identified by high-field EPR and DFT.

Project description:The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of biliverdin IXalpha to phycocyanobilin, the precursor of biliprotein chromophores found in phycobilisomes. It is known that catalysis proceeds via paramagnetic radical intermediates, but the structure of these intermediates and the transfer pathways for the four protons involved are not known. In this study, high-field electron paramagnetic resonance (EPR) spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of two PcyA mutants D105N from the cyanobacteria Synechocystis sp. PCC6803 and Nostoc sp. PCC7120 are examined. Detailed analysis of Synechocystis D105N mutant spectra at 130 and 406 GHz reveals a biliverdin radical with a very narrow g tensor with principal values 2.00359(5), 2.00341(5), and 2.00218(5). Using density-functional theory (DFT) computations to explore the possible protonation states of the biliverdin radical, it is shown that this g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated. This experimentally confirms the reaction mechanism recently proposed (Tu, et al. Biochemistry 2007, 46, 1484).

| S-EPMC2753389 | biostudies-literature

Fungal phytochrome chromophore biosynthesis at mitochondria

Project description:Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

| S-SCDT-EMBOJ-2021-108083 | biostudies-other

Fungal phytochrome chromophore biosynthesis at mitochondria.

Project description:Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

| S-EPMC8447599 | biostudies-literature

The phycocyanobilin chromophore of streptophyte algal phytochromes is synthesized by HY2.

Project description:Land plant phytochromes perceive red and far-red light to control growth and development, using the linear tetrapyrrole (bilin) chromophore phytochromobilin (PΦB). Phytochromes from streptophyte algae, sister species to land plants, instead use phycocyanobilin (PCB). PCB and PΦB are synthesized by different ferredoxin-dependent bilin reductases (FDBRs): PΦB is synthesized by HY2, whereas PCB is synthesized by PcyA. The pathway for PCB biosynthesis in streptophyte algae is unknown. We used phylogenetic analysis and heterologous reconstitution of bilin biosynthesis to investigate bilin biosynthesis in streptophyte algae. Phylogenetic results suggest that PcyA is present in chlorophytes and prasinophytes but absent in streptophytes. A system reconstituting bilin biosynthesis in Escherichia coli was modified to utilize HY2 from the streptophyte alga Klebsormidium flaccidum (KflaHY2). The resulting bilin was incorporated into model cyanobacterial photoreceptors and into phytochrome from the early-diverging streptophyte alga Mesostigma viride (MvirPHY1). All photoreceptors tested incorporate PCB rather than PΦB, indicating that KflaHY2 is sufficient for PCB synthesis without any other algal protein. MvirPHY1 exhibits a red-far-red photocycle similar to those seen in other streptophyte algal phytochromes. These results demonstrate that streptophyte algae use HY2 to synthesize PCB, consistent with the hypothesis that PΦB synthesis arose late in HY2 evolution.

| S-EPMC5388591 | biostudies-literature

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

Project description:Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IX? (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.

| S-EPMC6928796 | biostudies-literature

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases.

Project description:The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXalpha, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1-1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXalpha.

| S-EPMC26918 | biostudies-literature

Glyceraldehyde-3-phosphate ferredoxin oxidoreductase from Methanococcus maripaludis.

Project description:The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPOR(Pf)), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPOR(Mm)) was purified from Escherichia coli grown in minimal medium containing 100 muM sodium molybdate. In contrast, GAPOR(Mm) obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPOR(Mm) was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPOR(Mm) catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPOR(Mm) is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 muM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPOR(Mm) in M. maripaludis most likely involves only nonoptimal growth conditions.

| S-EPMC2168465 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data