The gun4 gene is essential for cyanobacterial porphyrin metabolism

Ycf53 is a hypothetical chloroplast open reading frame with similarity to the Arabidopsis nuclear gene GUN4. In plants, GUN4 is involved in tetrapyrrole biosynthesis. We demonstrate that one of the two Synechocystis sp. PCC 6803 ycf53 genes with similarity to GUN4 functions in chlorophyll (Chl) biosynthesis as well: cyanobacterial gun4 mutant cells exhibit lower Chl contents, accumulate protoporphyrin IX and show less activity not only of Mg chelatase but also of Fe chelatase. The possible role of Gun4 for the Mg as well as Fe porphyrin biosynthesis branches in Synechocystis sp. PCC 6803 is discussed.     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

The Chlorophyll Biosynthetic Enzyme Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase (Characterization and Partial Purification from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803)

A universal structural feature of chlorophyll molecules is the isocyclic ring. This ring is formed by the action of the enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, which catalyzes a complex reaction in which Mg-protoporphyrin IX monomethyl ester is converted to divinyl protochlorophyllide (also called Mg-2,4-divinylpheoporphyrin a5), with the participation of NADPH and O2. Cyclase activity was demonstrated in lysed Chlamydomonas reinhardtii chloroplasts and extracts of Synechocystis sp. PCC 6803. The yield of the reaction product was increased by the addition of catalase and ascorbate or isoascorbate to the incubation mixture. These compounds may act by preventing degradation of the tetrapyrroles by reactive oxygen species. Cyclase activity from C. reinhardtii was not inhibited by the flavoprotein inhibitor quinacrine or by the hemoprotein inhibitors CO, KCN, or NaN3. In contrast, cyclase activity in extracts of C. reinhardtii and Synechocystis sp. PCC 6803 was inhibited by chelators of Fe, suggesting that nonheme Fe is involved in the reaction. Cyclase in lysed C. reinhardtii chloroplasts was associated with the membranes, and attempts to further fractionate or solubilize the activity were unsuccessful. In contrast, cyclase in Synechocystis sp. PCC 6803 extracts could be separated into soluble and membrane components, both of which were required for reconstitution of activity. The membrane component retained activity after it was solubilized by the detergent n-octyl-[beta]-D-glucopyranoside in the presence of glycerol and Mg2+. The solubilized membrane component was purified further by dye-affinity and ion-exchange chromatography.     

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.     

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.     

Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase.

The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter. When a soluble extract from IPTG-induced E. coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E. coli expression vector. Soluble extract from an E. coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis. Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate. Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E. coli extracts. Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin.     

Functional characterization of sll0659 from Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that Sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types and mutants. In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cycilzation. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.     

Sll0254 (CrtL(diox)) is a bifunctional lycopene cyclase/dioxygenase in cyanobacteria producing myxoxanthophyll

Upon depletion of Sll0254 in Synechocystis sp. strain PCC 6803, cyclized carotenoids were replaced by linear, relatively hydrophilic carotenoids, and the amount of the two photosystems decreased greatly. Full segregants of the sll0254 deletion in Synechocystis were not obtained, implying that this gene is essential for survival, most likely to allow normal cell division. The N-terminal half of Sll0254 has limited similarity to the family of lycopene cyclases, has an additional dehydrogenase motif near the N terminus, and is followed by a Rieske 2Fe-2S center sequence signature. To test whether Sll0254 serves as a lycopene cyclase in Synechocystis, the corresponding gene was expressed in Escherichia coli strains that can produce lycopene or neurosporene. In the presence of Sll0254 these linear carotenoids were converted into cyclized, relatively hydrophilic pigments, with masses consistent with the introduction of two hydroxyl groups and with spectra indicative of only small changes in the number of conjugated double bonds. This suggests that Sll0254 catalyzes formation of oxygenated, cyclized carotenoids. We interpret the appearance of the hydroxyl groups in the carotenoids to be due to dioxygenase activity involving the Rieske 2Fe-2S center and the additional dehydrogenase domain. This dioxygenase activity is required in the myxoxanthophyll biosynthesis pathway, after or concomitant with cyclization on the other end of the molecule. We interpret Sll0254 to be a dual-function enzyme with both lycopene cyclase and dioxygenase activity and have named it CrtL(diox).     

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI). With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Deltaycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Deltaycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.     

Evidence for a stable association of Psb30 (Ycf12) with photosystem II core complex in the cyanobacterium Synechocystis sp. PCC 6803

Ycf12 (Psb30) is a small hydrophobic subunit of photosystem II (PS II) complexes found in the cyanobacterium, Thermosynechococcus elongatus. However, earlier intense proteomic analysis on the PS II complexes from the cyanobacterium, Synechocystis 6803, could not detect Psb30. In this work, we generated a mutant of Synechocystis 6803 in which a hexa-histidine tag was fused to the C-terminus of Synechocystis Psb30. The mutant accumulated fully functional PS II complexes. Purification of Psb30 by metal affinity chromatography from thylakoid extracts resulted in co-purification of an oxygen-evolving PS II complex with normal subunit composition. This result indicates that Psb30 is expressed and stably associated with the PS II complex in Synechocystis. The histidine-tagged Psb30 in the purified PS II complex was not detected by staining or anti-polyhistidine antibodies. We also generated a mutant in which ycf12 was disrupted. The mutant grew photosynthetically and showed no significant phenotype under moderate growth conditions. Purified PS II complexes from the disruptant showed an oxygen-evolving activity comparable to wild type under low irradiance. However, it showed a remarkably lower activity than wild type under high irradiance. Thus Psb30 is required for the efficient function of PS II complexes, particularly under high irradiance conditions.     

Identification of two homologous genes, chlAI and chlAII, that are differentially involved in isocyclic ring formation of chlorophyll a in the cyanobacterium Synechocystis sp. PCC 6803

The isocyclic ring (E-ring) is a common structural feature of chlorophylls. The E-ring is formed by two structurally unrelated Mg-protoporphyrin IX monomethylester (MPE) cyclase systems, oxygen-dependent (AcsF), and oxygen-independent (BchE) systems, which involve incorporation of an oxygen atom from molecular oxygen and water into the C-13(1) position of MPE, respectively. Which system operates in cyanobacteria that can thrive in a variety of anaerobic environments remains an open question. The cyanobacterium Synechocystis sp. PCC 6803 has two acsF-like genes, sll1214 (chlA(I)) and sll1874 (chlA(II)), and three bchE-like genes, slr0905, sll1242, and slr0309. Five mutants lacking one of these genes were isolated. The DeltachlA(I) mutant failed to grow under aerobic conditions with anomalous accumulation of a pigment with fluorescence emission peak at 595 nm, which was identified 3,8-divinyl MPE by high-performance liquid chromatography-mass spectrometry analysis. The growth defect of DeltachlA(I) was restored by the cultivation under oxygen-limited (micro-oxic) conditions. MPE accumulation was also detected in DeltachlA(II) grown under microoxic conditions, but not in any of the bchE mutants. The phenotype was consistent with the expression pattern of two chlA genes: chlA(II) was induced under micro-oxic conditions in contrast to the constitutive expression of chlA(I). These findings suggested that ChlA(I) is the sole MPE cyclase system under aerobic conditions and that the induced ChlA(II) operates together with ChlA(I) under micro-oxic conditions. In addition, the accumulation of 3,8-divinyl MPE in the DeltachlA mutants suggested that the reduction of 8-vinyl group occurs after the formation of E-ring in Synechocystis sp. PCC 6803.     

MPEC: an important gene in the chlorophyll biosynthesis pathway in photosynthetic organisms

One of the least understood enzymatic steps in chlorophyll biosynthesis is the formation of isocyclic ring, which is catalyzed by the Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase that is involved in the conversion of MgPME to protochlorophyllide. Several genes encoding part of this enzyme have been identified and functional analysis of them has been performed. The enzyme plays important roles in higher plants and photosynthetic bacteria. The review focuses on the current knowledge of MgPME cyclase coding genes, with emphasis on their organization, expression pattern, and functional analysis obtained from mutants.     

Late assembly steps and dynamics of the cyanobacterial photosystem I

The dynamics of photosystem I assembly in cyanobacteria have been addressed using in vivo pulse-chase labeling of Synechocystis sp. PCC 6803 proteins in combination with blue native polyacrylamide gel electrophoresis. The analyses indicate the existence of three different monomeric photosystem I complexes and also the high stability of photosystem I trimers. We show that in addition to a complete photosystem I monomer, containing all 11 subunits, we detected a PsaK-less monomer and a short-lived PsaL/PsaK-less complex. The latter two monomers were missing in the ycf37 mutant of Synechocystis sp. PCC 6803 that accumulates also less trimers. Pulse-chase experiments suggest that the three monomeric complexes have different functions in the biogenesis of the trimer. Based on these findings we propose a model where PsaK is incorporated in the latest step of photosystem I assembly. The PsaK-less photosystem I monomer may represent an intermediate complex that is important for the exchange of the two PsaK variants during high light acclimation. Implications of the presented data with respect to Ycf37 function are discussed.     

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-l-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.     

Synechocystis strain PCC 6803 cya2, a prokaryotic gene that encodes a guanylyl cyclase

Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.     

Modulation of chlorophyll biosynthesis by water stress in rice seedlings during chloroplast biogenesis*

To understand the impact of water stress on the greening process, water stress was applied to 6-day-old etiolated seedlings of a drought-sensitive cultivar of rice (Oryza sativa), Pusa Basmati-1 by immersing their roots in 40 mm polyethylene glycol (PEG) 6000 (-0.69 MPa) or 50 mm PEG 6000 (-1.03 MPa) dissolved in half-strength Murashige and Skoog (MS)-nutrient-solution, 16 h prior to transfer to cool-white-fluorescent + incandescent light. Chlorophyll (Chl) accumulation substantially declined in developing water-stressed seedlings. Reduced Chl synthesis was due to decreased accumulation of chlorophyll biosynthetic intermediates, that is, glutamate-1-semialdehyde (GSA), 5-aminolevulinic acid, Mg-protoporphyrin IX monomethylester and protochlorophyllide. Although 5-aminolevulinic acid synthesis decreased, the gene expression and protein abundance of the enzyme responsible for its synthesis, GSA aminotransferase, increased, suggesting its crucial role in the greening process in stressful environment. The biochemical activities of Chl biosynthetic enzymes, that is, 5-aminolevulinic acid dehydratase, porphobilinogen deaminase, coproporphyrinogen III oxidase, porphyrinogen IX oxidase, Mg-chelatase and protochlorophyllide oxidoreductase, were down-regulated due to their reduced protein abundance/gene expression in water-stressed seedlings. Down-regulation of protochlorophyllide oxidoreductase resulted in impaired Shibata shift. Our results demonstrate that reduced synthesis of early intermediates, that is, GSA and 5-aminolevulinic acid, could modulate the gene expression of later enzymes of Chl biosynthesis pathway.     

Heterologous expression of the bchM gene product from Rhodobacter capsulatus and demonstration that it encodes S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase.

The bacteriochlorophyll biosynthesis gene, bchM, from Rhodobacter capsulatus was previously believed to code for a polypeptide involved in formation of the cyclopentone ring of protochlorophyllide from Mg-protoporphyrin IX monomethyl ester. In this study, R. capsulatus bchM was expressed in Escherichia coli and the gene product was subsequently demonstrated by enzymatic analysis to catalyze methylation of Mg-protoporphyrin IX to form Mg-protoporphyrin IX monomethyl ester. Activity required the substrates Mg-protoporphyrin IX and S-adenosyl-L-methionine. 14C-labeled product was formed in incubations containing 14C-methyl-labeled S-adenosyl-L-methionine. On the basis of these and previous results, we also conclude that the bchH gene, which was previously reported to code for Mg-protoporphyrin IX methyltransferase, is most likely involved in the Mg chelation step.