scholarly journals Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1157
Author(s):  
David Stuart ◽  
Malin Sandström ◽  
Helmy M. Youssef ◽  
Shakhira Zakhrabekova ◽  
Poul Erik Jensen ◽  
...  
Keyword(s):  
Protoporphyrin Ix ◽  
Photosynthetic Electron Transport ◽  
In Vitro Assays ◽  
Light Conditions ◽  
Hordeum Vulgare L ◽  
Transport Chain ◽  
Additional Protein ◽  
Membrane Bound ◽  
Monomethyl Ester

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.

Plastoquinone-9 biosynthesis in cyanobacteria differs from that in plants and involves a novel 4-hydroxybenzoate solanesyltransferase

2012 ◽  
Vol 442 (3) ◽  
pp. 621-629 ◽  
Author(s):  
Radin Sadre ◽  
Christian Pfaff ◽  
Stephan Buchkremer
Keyword(s):  
Biosynthetic Pathway ◽  
In Vitro Assays ◽  
Transport Chain ◽  
In Vivo Labelling ◽  
Membrane Bound ◽  
Transformation Processes ◽  
Synechocystis Sp

PQ-9 (plastoquinone-9) has a central role in energy transformation processes in cyanobacteria by mediating electron transfer in both the photosynthetic as well as the respiratory electron transport chain. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. We identified 4-hydroxybenzoate as being the aromatic precursor for PQ-9 in Synechocystis sp. PCC6803, and in the present paper we report on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an Escherichia coli mutant with a defect in ubiquinone biosynthesis, and in vitro assays using the recombinant as well as the native enzyme. Although Slr0926 was highly specific for the prenyl acceptor substrate 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate, but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferases catalysing prenylation in the course of ubiquinone biosynthesis.

The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria

2010 ◽  
Vol 431 (1) ◽  
pp. 135-140 ◽  
Author(s):  
Mayumi Horiuchi ◽  
Kinu Nakamura ◽  
Kouji Kojima ◽  
Yoshitaka Nishiyama ◽  
Wakako Hatakeyama ◽  
...  
Keyword(s):  
Electron Transport ◽  
Conformational Change ◽  
Photosynthetic Electron Transport ◽  
Transcript Level ◽  
High Light ◽  
Light Conditions ◽  
Transport Chain ◽  
Acclimation Response ◽  
Vitro Analysis

The redox state of the photosynthetic electron transport chain acts as a critical sensing mechanism by regulating the transcription of key genes involved in the acclimation response to a change in the environment. In the present study we show that the small LuxR-type regulator PedR interacts with Trx (thioredoxin) to achieve photosynthetic electron-transport-dependent transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. TrxM, an isoform of Trx, was isolated as an interacting factor of PedR by pull-down assays. In vitro analysis revealed that the intermolecular disulfide bond formed between Cys80 residues of the PedR homodimer was reduced by both TrxM and TrxX. It has been shown previously that, although PedR is active under low-light conditions, it becomes transiently inactivated following a shift to high-light conditions, with a concomitant conformational change [Nakamura and Hihara (2006) J. Biol. Chem. 281, 36758–36766]. In the present study, we found that the conformational change of PedR and the change in the transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin–Trx reductase or NADPH–Trx reductase were exposed to high levels of light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions.

scholarly journals The Role of Selected Wavelengths of Light in the Activity of Photosystem II in Gloeobacter violaceus

2021 ◽  
Vol 22 (8) ◽  
pp. 4021
Author(s):  
Monika Kula-Maximenko ◽  
Kamil Jan Zieliński ◽  
Ireneusz Ślesak
Keyword(s):  
Photosystem Ii ◽  
Spectral Composition ◽  
Photosynthetic Electron Transport ◽  
Metabolic Energy ◽  
Light Conditions ◽  
Gloeobacter Violaceus ◽  
Transport Chain ◽  
Cyanobacterial Culture ◽  
Photosynthetic Antennas

Gloeobacter violaceus is a cyanobacteria species with a lack of thylakoids, while photosynthetic antennas, i.e., phycobilisomes (PBSs), photosystem II (PSII), and I (PSI), are located in the cytoplasmic membrane. We verified the hypothesis that blue–red (BR) light supplemented with a far-red (FR), ultraviolet A (UVA), and green (G) light can affect the photosynthetic electron transport chain in PSII and explain the differences in the growth of the G. violaceus culture. The cyanobacteria were cultured under different light conditions. The largest increase in G. violaceus biomass was observed only under BR + FR and BR + G light. Moreover, the shape of the G. violaceus cells was modified by the spectrum with the addition of G light. Furthermore, it was found that both the spectral composition of light and age of the cyanobacterial culture affect the different content of phycobiliproteins in the photosynthetic antennas (PBS). Most likely, in cells grown under light conditions with the addition of FR and G light, the average antenna size increased due to the inactivation of some reaction centers in PSII. Moreover, the role of PSI and gloeorhodopsin as supplementary sources of metabolic energy in the G. violaceus growth is discussed.

The atypical kinase ABC1K1 interacts with the EXECUTER pathway to promote chloroplast biogenesis.

2021 ◽  
Author(s):  
Joy Collombat ◽  
Thibaut Pralon ◽  
Jenny Pego Magalhaes ◽  
Sarah Rottet ◽  
Brigitte Ksas ◽  
...  
Keyword(s):  
Electron Transport Chain ◽  
Red Light ◽  
Photosynthetic Electron Transport ◽  
Chloroplast Biogenesis ◽  
Light Conditions ◽  
Photosynthetic Electron Transport Chain ◽  
Transport Chain ◽  
Dependent Signal ◽  
Photosynthetic Mutants

Abstract Multiple chloroplast-to-nucleus signaling pathways contribute to the regulation of chloroplast biogenesis during plant greening. Here, we provide evidence for the direct implication of the atypical kinase ABC1K1. ABC1K1 is required for sufficient plastoquinone (PQ) allocation to the photosynthetic electron transport chain. Unexpectedly, mutation of abc1k1 suppresses greening and results in pale cotyledons under red light. This phenotype was not observed in other photosynthetic mutants and points to a specific signaling defect. Under red light, abc1k1 accumulated EXECUTER1 (EX1), a trigger of singlet oxygen (1O2) signaling. Consistent with the role of the FTSH metalloprotease in chloroplast biogenesis and EX1 degradation, the ftsh2 mutant var2, mimicked the greening defect of abc1k1 and accumulated EX1 under red light. We propose that this novel ABC1K1-dependent signal is required for chloroplast biogenesis to progress in challenging light conditions.

scholarly journals Characterizing membrane anchoring of leaf-form ferredoxin-NADP+ oxidoreductase in rice

2021 ◽  
Author(s):  
xiaowen da ◽  
jiangfan guo ◽  
peng yan ◽  
Chao Yang ◽  
Hongfei Zhao ◽  
...  
Keyword(s):  
Thylakoid Membrane ◽  
Photosynthetic Electron Transport ◽  
Light Conditions ◽  
Membrane Anchor ◽  
Chloroplast Membranes ◽  
Leaf Form ◽  
Photosynthetic Electron Transport Chain ◽  
Transport Chain ◽  
Chloroplast Stroma ◽  
Membrane Anchoring

Leaf-form ferredoxin-NADP+ oxidoreductases (LFNRs) function in the last step of the photosynthetic electron transport chain, exist as soluble proteins in the chloroplast stroma, and are weakly associated with thylakoids or tightly anchored to chloroplast membranes. Arabidopsis thaliana has two LFNRs, and the chloroplast proteins AtTROL (THYLAKOID RHODANESE-LIKE PROTEIN) and AtTIC62 (62-kDa SUBUNIT OF TRANSLOCON OF INNER CHLOROPLAST MEMBRANE) participate in anchoring AtLFNRs to the thylakoid membrane. By contrast, the membrane anchoring mechanism of rice (Oryza sativa) LFNRs has not been elucidated. Here, we investigated the membrane-anchoring mechanism of LFNRs and its physiological roles in rice. We characterized the rice protein OsTROL1 based on its homology to AtTROL and showed that OsTROL1 is also a thylakoid membrane anchor and its loss led to a compensatory increase in OsTIC62. Moreover, OsLFNR1 attachment through a membrane anchor depends on OsLFNR2, unlike their Arabidopsis counterparts. In addition, OsTIC62 was more highly expressed in rice under dark than under light conditions, consistent with the increased membrane binding of OsLFNR in the dark. Moreover, we observed reciprocal stabilization between OsLFNRs and their membrane anchors. Therefore, our study sheds light on the mechanisms anchoring LFNRs to membranes in rice and highlights differences with Arabidopsis

scholarly journals Structure-Based Design of Nucleoside-Derived Analogues as Sulfotransferase Inhibitors

2019 ◽  
Author(s):  
Neil Kershaw ◽  
Dominic Byrne ◽  
Hollie Parsons ◽  
Neil G Berry ◽  
David Fernig ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Methyl Esters ◽  
Cell Signalling ◽  
In Vitro Assays ◽  
Protein Protein Interactions ◽  
Tyrosine Sulfation ◽  
Post Translational Modifications ◽  
Relative Specificity ◽  
Membrane Bound

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

scholarly journals Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls

2020 ◽  
Vol 477 (23) ◽  
pp. 4635-4654
Author(s):  
Milan Wiesselmann ◽  
Stefanie Hebecker ◽  
José M. Borrero-de Acuña ◽  
Manfred Nimtz ◽  
David Bollivar ◽  
...  
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Homology Model ◽  
Subcellular Fractionation ◽  
Site Directed Mutagenesis ◽  
Bioinformatics Analyses ◽  
Monomethyl Ester

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

HPLC and in vivo Spectrophotometric Detection of Porphyrins in Plant Tissues Treated with Porphyrinogenic Herbicides

1993 ◽  
Vol 48 (3-4) ◽  
pp. 317-325 ◽  
Author(s):  
Stephen O. Duke ◽  
Mary V. Duke ◽  
Hee Jae Lee
Keyword(s):  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Difference Spectrum ◽  
Plant Tissues ◽  
Cucumber Cotyledon ◽  
The Difference ◽  
Dynamic Damage ◽  
Monomethyl Ester

Abstract Protoporphyrinogen oxidase (Protox) inhibitors and other compounds which block or stimulate the porphyrin pathway can cause sufficient levels of porphyrins to accumulate in plant tissues for severe photo dynamic damage to occur. The gross symptomology for all of these por­phyrinogenic herbicides is similar. Porphyrin accumulation induced by three porphyrinogenic herbicides acifluorfen (AF), δ-aminolevulinic acid (ALA), and 2,2′-dipyridyl (DY) was determined by in vivo spectrophotometry and HPLC methods. The averaged in vivo difference spec­tra between untreated and AF-treated (30 μᴍ for 20 h in darkness) yellow cucumber cotyledon discs approximated the absorption spectra of protoporphyrin IX (Proto IX). There was also an enhanced peak near 503 nm. Treatment of cotyledon discs with ALA alone generated a difference spectrum of protochlorophyllide (PChlide) in combination with Mg -Proto IX or Mg-Proto IX monomethyl ester (Mg-Proto IX ME). With ALA and AF in combination , the PChlide and Mg-Proto IX portions of the difference spectrum were reduced and the Proto IX peak and peak near 503 nm were increased. DY treatment yielded a difference spectrum with peaks approximating those of Proto IX and Mg-Proto IX ME , along with a peak near 503 nm . The presence of all porphyrins detected by in vivo spectrophotometry except for the 503 nm peak was confirmed with HPLC . Proto IX monomethyl ester was found by HPLC to be espe­cially elevated in treatments with AF. The in vivo 503 nm peak and in vitro studies with Protox-containing barley etioplast preparations suggest that p rototetrahydroporphyrin IX (an oxida­tion state intermediate between protoporphyrinogen IX and Proto IX) may accumulate under some conditions. These data demonstrate that rapid in vivo spectrophotometric studies can provide much of the qualitative results of HPLC studies and can confirm that in vitro results correspond with the in vivo situation.

scholarly journals Structure-Based Design of Nucleoside-Derived Analogues as Sulfotransferase Inhibitors

2019 ◽  
Author(s):  
Neil Kershaw ◽  
Dominic Byrne ◽  
Hollie Parsons ◽  
Neil G Berry ◽  
David Fernig ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Methyl Esters ◽  
Cell Signalling ◽  
In Vitro Assays ◽  
Protein Protein Interactions ◽  
Tyrosine Sulfation ◽  
Post Translational Modifications ◽  
Relative Specificity ◽  
Membrane Bound

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

Sign in / Sign up

Export Citation Format

Share Document