Structure of GUN4 fromChlamydomonas reinhardtii

The genomes uncoupled 4 (GUN4) protein stimulates chlorophyll biosynthesis by increasing the activity of Mg-chelatase, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway. One of the roles of GUN4 is in binding PPIX and Mg-PPIX. In eukaryotes, GUN4 also participates in plastid-to-nucleus signalling, although the mechanism for this is unclear. Here, the first crystal structure of a eukaryotic GUN4, fromChlamydomonas reinhardtii, is presented. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational divergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to the understanding of how GUN4 binds PPIX or Mg-PPIX.

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Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI–BchD complex

Biochemical Journal ◽

10.1042/bj3370243 ◽

1999 ◽

Vol 337 (2) ◽

pp. 243-251 ◽

Cited By ~ 3

Author(s):

Lucien C. D. GIBSON ◽

Poul Erik JENSEN ◽

C. Neil HUNTER

Keyword(s):

Rhodobacter Sphaeroides ◽

Chlorophyll Biosynthesis ◽

Gel Filtration ◽

Protoporphyrin Ix ◽

Photosynthetic Bacterium ◽

Affinity Tags ◽

Magnesium Chelatase ◽

Highly Active ◽

Mg Chelatase

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchDand bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.

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Role of the antioxidant system in the regulation of the chlorophyll biosynthesis pathway in the vascular plant Cucumis sativus

Functional Plant Biology ◽

10.1071/fp16393 ◽

2018 ◽

Vol 45 (4) ◽

pp. 464

Author(s):

Aarti Dhepe ◽

Komal Joshi

Keyword(s):

Cucumis Sativus ◽

Antioxidant System ◽

Chlorophyll Biosynthesis ◽

Vascular Plant ◽

Protoporphyrin Ix ◽

Thioredoxin Reductase ◽

Ros Generation ◽

Biosynthesis Pathway ◽

Cucumis Sativus L

In this study, the role of the antioxidant system has been examined in the regulation of the chlorophyll biosynthesis pathway in the vascular plant Cucumis sativus L. To generate reactive oxygen species (ROS), etiolated (E) and green (G) cucumber cotyledons were treated with methyl viologen (MV) or were exposed to high light (HL, 400–500 µE m–2 s–1). ROS generation was confirmed by measuring proline and H2O2 concentrations. With the effects of the MV- and HL-induced oxidative stress, it was observed that the chlorophyll biosynthesis pathway was severely affected in the HL-treated etiolated cotyledons (E-HL), MV-treated etiolated cotyledons (E-MV) and in MV-treated green cotyledons (G-MV) at 5-amino levulinic acid (ALA) as well as at protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester levels. The antioxidant assays conducted showed that the ascorbate peroxidase (APX) activity had decreased in the E-HL, E-MV and G-MV cotyledons along with the levels of ascorbate and lutein. A decrease in the NADPH-dependent thioredoxin reductase (NTRC) was also observed in the MV-treated cotyledons with a significant impairment of the catalase activity in the E-HL cotyledons. Conversely, in the HL-treated green i.e. G-HL cotyledons, where the accumulation of H2O2 and the inhibition of chlorophyll biosynthesis were not observed, an increase in the levels of APX, NTRC, peroxiredoxin, ascorbate, glutathione and lutein was noted. Thus, the results obtained suggested that the antioxidant system could influence the flow of the chlorophyll biosynthesis pathway through maintaining the levels of H2O2.

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LTS3 gene controls light-independent chlorophyll biosynthesis in green algae Chlamydomonas reinhardtii

Ecological genetics ◽

10.17816/ecogen8235-44 ◽

2010 ◽

Vol 8 (2) ◽

pp. 35-44

Author(s):

Elena M Chekunova ◽

Natalya V Savelieva

Keyword(s):

Chlamydomonas Reinhardtii ◽

Positional Cloning ◽

Chlorophyll Biosynthesis ◽

Plant Cells ◽

Expression Of Genes ◽

Gata Transcription Factor ◽

Pigment Contents ◽

Genes Encoding ◽

Activity Of Enzymes ◽

Mg Chelatase

The genetic control of light-independent chlorophyll biosynthesis in plant cells has been investigated using Chlamydomonas reinhardtii Lts3-mutants defective in dark chlorophyll biosynthesis on the stage before protochlorophyllide to chlorophyllide conversion. In heterotrophic conditions the mutants are unable to synthesize chlorophyll and accumulate protoporphyrins, after illumination they are greening. The mutants were tested for pigment contents, activity of enzymes and expression of the genes, encoding these enzymes. The LTS3 gene has been identified by positional cloning, and the predicted LTS3 protein appeared to be a GATA transcription factor, which activate the expression of genes encoded chlorophyll biosynthesis enzymes: Mg-chelatase and glutamate 1-semialdehyde aminotransferase in the dark, and possibly, important for adaptation of plant cells for autotrophic conditions.

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Mg-Protoporphyrin IX and Heme Control HEMA, the Gene Encoding the First Specific Step of Tetrapyrrole Biosynthesis, in Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.4.10.1620-1628.2005 ◽

2005 ◽

Vol 4 (10) ◽

pp. 1620-1628 ◽

Cited By ~ 19

Author(s):

Zinaida Vasileuskaya ◽

Ulrike Oster ◽

Christoph F. Beck

Keyword(s):

Chlamydomonas Reinhardtii ◽

Posttranscriptional Regulation ◽

Higher Plants ◽

Chlorophyll Biosynthesis ◽

Single Gene ◽

Protoporphyrin Ix ◽

Tetrapyrrole Biosynthesis ◽

Gene Encoding ◽

Mrna Accumulation ◽

Specific Step

ABSTRACT HEMA encodes glutamyl-tRNA reductase (GluTR), which catalyzes the first step specific for tetrapyrrole biosynthesis in plants, archaea, and most eubacteria. In higher plants, GluTR is feedback inhibited by heme and intermediates of chlorophyll biosynthesis. It plays a key role in controlling flux through the tetrapyrrole biosynthetic pathway. This enzyme, which in Chlamydomonas reinhardtii is encoded by a single gene (HEMA), exhibits homology to GluTRs of higher plants and cyanobacteria. HEMA mRNA accumulation was inducible not only by light but also by treatment of dark-adapted cells with Mg-protoporphyrin IX (MgProto) or hemin. The specificity of these tetrapyrroles as inducers was demonstrated by the absence of induction observed upon the feeding of protoporphyrin IX, the precursor of both heme and MgProto, or chlorophyllide. The HEMA mRNA accumulation following treatment of cells with light and hemin was accompanied by increased amounts of GluTR. However, the feeding of MgProto did not suggest a role for Mg-tetrapyrroles in posttranscriptional regulation. The induction by light but not that by the tetrapyrroles was prevented by inhibition of cytoplasmic protein synthesis. Since MgProto is synthesized exclusively in plastids and heme is synthesized in plastids and mitochondria, the data suggest a role of these compounds as organellar signals that control expression of the nuclear HEMA gene.

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Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

F1000Research ◽

10.12688/f1000research.2-138.v1 ◽

2013 ◽

Vol 2 ◽

pp. 138 ◽

Cited By ~ 1

Author(s):

Phillip B Grovenstein ◽

Darryel A Wilson ◽

Cameron G Lennox ◽

Katherine P Smith ◽

Alisha A Contractor ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Insertional Mutagenesis ◽

Chlorophyll Biosynthesis ◽

Protoporphyrin Ix ◽

Model Organism ◽

Oxygenic Photosynthesis ◽

Functional Roles ◽

Protein Levels ◽

In The Wild ◽

Chl Biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis and chloroplast to nucleus retrograde signaling in Chlamydomonas, which has never been studied before.

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Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

F1000Research ◽

10.12688/f1000research.2-138.v2 ◽

2013 ◽

Vol 2 ◽

pp. 138

Author(s):

Phillip B Grovenstein ◽

Darryel A Wilson ◽

Cameron G Lennox ◽

Katherine P Smith ◽

Alisha A Contractor ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Insertional Mutagenesis ◽

Chlorophyll Biosynthesis ◽

Protoporphyrin Ix ◽

Model Organism ◽

Oxygenic Photosynthesis ◽

Functional Roles ◽

Protein Levels ◽

In The Wild ◽

Chl Biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.

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Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713034275 ◽

2014 ◽

Vol 70 (4) ◽

pp. 1166-1172 ◽

Cited By ~ 5

Author(s):

Michael E. Webb ◽

Briony A. Yorke ◽

Tom Kershaw ◽

Sarah Lovelock ◽

Carina M. C. Lobley ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Site Directed Mutagenesis ◽

Intramolecular Rearrangement ◽

Directed Mutagenesis ◽

Vitamin B ◽

Biosynthesis Pathway ◽

Serine Residue ◽

Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

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The Crystal Structure of the Periplasmic Domain of the Escherichia coli Membrane Protein Insertase YidC Contains a Substrate Binding Cleft

Journal of Biological Chemistry ◽

10.1074/jbc.m710493200 ◽

2008 ◽

Vol 283 (14) ◽

pp. 9350-9358 ◽

Cited By ~ 39

Author(s):

Stephanie Ravaud ◽

Goran Stjepanovic ◽

Klemens Wild ◽

Irmgard Sinning

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Membrane Protein ◽

Substrate Binding ◽

Periplasmic Domain ◽

Binding Cleft

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Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

Biochemical Journal ◽

10.1042/bj3590065 ◽

2001 ◽

Vol 359 (1) ◽

pp. 65-75 ◽

Cited By ~ 25

Author(s):

Valeria MENCHISE ◽

Catherine CORBIER ◽

Claude DIDIERJEAN ◽

Michele SAVIANO ◽

Ettore BENEDETTI ◽

...

Keyword(s):

Crystal Structure ◽

Proton Transfer ◽

Chlamydomonas Reinhardtii ◽

Catalytic Mechanism ◽

Structural Data ◽

Careful Analysis ◽

Wild Type ◽

Thioredoxin H ◽

Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

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