Bilin-dependent regulation of chlorophyll biosynthesis by GUN4

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii. A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment.

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Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.2.c703 ◽

1997 ◽

Vol 273 (2) ◽

pp. C703-C709 ◽

Cited By ~ 31

Author(s):

R. Henningsson ◽

P. Alm ◽

I. Lundquist

Keyword(s):

Enzymatic Activity ◽

Insulin Release ◽

Islets Of Langerhans ◽

Heme Oxygenase ◽

High Glucose ◽

Protoporphyrin Ix ◽

Regulatory Function ◽

Glucagon Release ◽

Hormone Release ◽

Islet Hormone

Recent observations suggest that carbon monoxide (CO) may serve as a neuroendocrine modulator in hypothalamus. Here we provide evidence, for the first time, that the islets of Langerhans contain the constitutive isoform of the CO-producing enzyme heme oxygenase (HO-2), the activity of which was found to modulate islet hormone release. Most insulin and glucagon cells in the rat endocrine pancreas expressed strong immunoreactivity for HO-2. In the exocrine parenchyma, scattered HO-2-positive ganglionic cell bodies were occasionally observed. Furthermore, Western blot analysis revealed the presence of HO-2 in isolated islets but not in acinar cells. Islet homogenates displayed a comparatively high HO-2 enzymatic activity measured as CO formation (approximately 600 pmol CO.min-1.mg islet protein-1). This HO-2 enzymatic activity was greatly suppressed by zincprotoporphyrin-IX (ZnPP-IX), a recognized inhibitor of HO activity. Neither ZnPP-IX nor the HO activator, hemin, influenced basal insulin release from isolated rat islets at low (1 mM) glucose. However, glucagon release at 1 mM glucose was increased by hemin and inhibited by ZnPP-IX. The hemin-induced increase in glucagon secretion was abolished by ZnPP-IX. Furthermore, a series of experiments at high glucose (16.7 mM) revealed that hemin induced a dose-dependent potentiation of glucose-stimulated insulin release. Moreover, glucose-induced insulin release was dose-dependently suppressed by ZnPP-IX but unaffected by protoporphyrin-IX, a compound known not to influence HO-2 activity in other tissues. Similarly, glucagon release at high glucose was dose-dependently increased by hemin and suppressed by ZnPP-IX. Finally, the hemin-induced increase in islet hormone release at high glucose was totally abolished by ZnPP-IX. The data strongly suggest that CO production positively modulates both glucagon and insulin secretion. We propose that CO may serve as a novel messenger molecule within the islets of Langerhans.

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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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Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation

Biochemical Journal ◽

10.1042/bj3520435 ◽

2000 ◽

Vol 352 (2) ◽

pp. 435-441 ◽

Cited By ~ 18

Author(s):

Poul E. JENSEN ◽

James D. REID ◽

C. Neil HUNTER

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Chlorophyll Biosynthesis ◽

Protoporphyrin Ix ◽

Cysteine Residue ◽

Enzyme Inactivation ◽

Cysteine Residues ◽

Magnesium Chelatase ◽

Synechocystis Pcc6803 ◽

Chelation Reaction

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg2+ chelatase, such as the ATPase activity of ChlI, and the formation of ChlI–ChlD–MgATP and ChlH–protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg2+ chelatase activity, and the formation of the ChlI–ChlD–ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI–ChlD–MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg2+ ions. The simplest explanation for this is that in an intact Mg2+ chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI–MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

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S-Adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids

Biochemical Journal ◽

10.1042/bj20070284 ◽

2007 ◽

Vol 406 (3) ◽

pp. 469-478 ◽

Cited By ~ 6

Author(s):

Artur Sawicki ◽

Robert D. Willows

Keyword(s):

Enzymatic Activity ◽

Inclusion Bodies ◽

Rhodobacter Capsulatus ◽

Protoporphyrin Ix ◽

Size Exclusion ◽

Zinc Protoporphyrin ◽

Sequential Reaction ◽

Magnesium Chelatase ◽

Heat Stable ◽

Exclusion Chromatography

The enzyme BchM (S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase) from Rhodobacter capsulatus catalyses an intermediate reaction in the bacteriochlorophyll biosynthetic pathway. Overexpression of His6-tagged protein in Escherichia coli resulted in the majority of polypeptide existing as inclusion bodies. Purification from inclusion bodies was performed using metal-affinity chromatography after an elaborate wash step involving surfactant polysorbate-20. Initial enzymatic assays involved an in situ generation of S-adenosyl-L-methionine substrate using a crude preparation of S-adenosyl-L-methionine synthetase and this resulted in higher enzymatic activity compared with commercial S-adenosyl-L-methionine. A heat-stable stimulatory component present in the S-adenosyl-L-methionine synthetase was found to be a phospholipid, which increased enzymatic activity 3–4-fold. Purified phospholipids also stabilized enzymatic activity and caused a disaggregation of the protein to lower molecular mass forms, which ranged from monomeric to multimeric species as determined by size-exclusion chromatography. There was no stimulatory effect observed with magnesium–chelatase subunits on methyltransferase activity using His–BchM that had been stabilized with phospholipids. Substrate specificity of the enzyme was limited to 5-co-ordinate square-pyramidal metalloporphyrins, with magnesium-protoporphyrin IX being the superior substrate followed by zinc-protoporphyrin IX and magnesium-deuteroporphyrin. Kinetic analysis indicated a random sequential reaction mechanism. Three non-substrate metalloporphyrins acted as inhibitors with different modes of inhibition exhibited with manganese III-protoporphyrin IX (non-competitive or uncompetitive) compared with cobalt II-protoporphyrin IX (competitive).

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The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Biochemical Journal ◽

10.1042/bcj20190095 ◽

2019 ◽

Vol 476 (13) ◽

pp. 1875-1887 ◽

Cited By ~ 7

Author(s):

David A. Farmer ◽

Amanda A. Brindley ◽

Andrew Hitchcock ◽

Philip J. Jackson ◽

Bethany Johnson ◽

...

Keyword(s):

Metal Ion ◽

Chlorophyll Biosynthesis ◽

Protoporphyrin Ix ◽

Catalytic Core ◽

Magnesium Chelatase ◽

Aaa Atpases ◽

Hydrolysis Of ◽

Chemical Cross Linking

Abstract Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2−-dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (Kd 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD–H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL–MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.

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The protective properties of synthetic porphyrin tin complexes in toxic hyperbilirubinemia

Journal of Porphyrins and Phthalocyanines ◽

10.1002/(sici)1099-1409(200004/05)4:3<243::aid-jpp201>3.0.co;2-d ◽

2000 ◽

Vol 04 (03) ◽

pp. 243-247 ◽

Cited By ~ 9

Author(s):

T. O. PHILIPPOVA ◽

B. N. GALKIN ◽

N. YA. GOLOVENKO ◽

Z. I. ZHILINA ◽

S. V. VODZINSKII

Keyword(s):

Heme Oxygenase ◽

Serum Bilirubin ◽

Protoporphyrin Ix ◽

Serum Bilirubin Level ◽

Oxygenase Activity ◽

Tetraphenyl Porphyrin ◽

Tin Complexes ◽

Cytochrome P 450

Tin complexes of meso-substituted synthetic porphyrins, namely Sn 4+-meso-tetraphenyl- porphyrin ( Sn - TPP ) and Sn 4+-meso-tetrakis(N-methyl-3-pyridyl)porphyrin tetratosylate ( Sn - TMe -3- PyP ), efficiently decrease the serum bilirubin level when injected subcutaneously at a dose of 100 μM kg−1 body weight into mice. These compounds are active during hyperbilirubinemia, induced by phenylhydrazine, hemin and tetrachloromethane, and also during autoimmune hemolytic anemia. In the latter case a decrease in serum bilirubin content was observed, as well as a decrease in the amount of blood reticulocytes which reflects a milder course of the disease. The Sn complexes under study induce, in vivo, cytochrome P-450, inhibit microsomal heme oxygenase and decrease the intensity of lipid peroxidation. At the same time, in vitro the hepatic and splenic heme oxygenase activity is blocked only when a 0.1 μM concentration of Sn - TMe -3- PyP or Sn -protoporphyrin IX is added to the incubation mixture. Sn - TPP does not affect the activity of this enzyme in vitro.

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Effect of glucose and oxygen deprivation on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00234.2003 ◽

2003 ◽

Vol 285 (6) ◽

pp. R1453-R1460 ◽

Cited By ~ 11

Author(s):

S. D. Appleton ◽

G. E. Lash ◽

G. S. Marks ◽

K. Nakatsu ◽

J. F. Brien ◽

...

Keyword(s):

Enzymatic Activity ◽

Protein Content ◽

Heme Oxygenase ◽

Glucose Concentration ◽

Placental Tissue ◽

Glucose Deprivation ◽

First Trimester ◽

Trophoblast Cells ◽

Chorionic Villi ◽

Low Glucose

Although hypoxia induces heme oxygenase (HO)-1 mRNA and protein expression in many cell types, recent studies in our laboratory using human placental tissue have shown that a preexposure to hypoxia does not affect subsequent HO enzymatic activity for optimized assay conditions (20% O2; 0.5 mM NADPH; 25 μM methemalbumin) or HO-1 protein content. One of the consequences of impaired blood flow is glucose deprivation, which has been shown to be an inducer of HO-1 expression in HepG2 hepatoma cells. The objective of the present study was to test the effects of a 24-h preexposure to glucose-deprived medium, in 0.5 or 20% O2, on HO protein content and enzymatic activity in isolated chorionic villi and immortalized HTR-8/SVneo first-trimester trophoblast cells. HO protein content was determined by Western blot analysis, and microsomal HO enzymatic activity was measured by assessment of the rate of CO formation. HO enzymatic activity was increased ( P < 0.05) in both placental models after 24-h preexposure to glucose-deficient medium in 0.5 or 20% O2. Preexposure (24 h) in a combination of low O2 and low glucose concentrations decreased the protein content of the HO-1 isoform by 59.6% ( P < 0.05), whereas preexposure (24 h) to low glucose concentration alone increased HO-2 content by 28.2% in chorionic villi explants ( P < 0.05). In this preparation, HO enzymatic activity correlated with HO-2 protein content ( r = 0.825). However, there was no correlation between HO-2 protein content and HO enzymatic activity in HTR-8/SVneo trophoblast cells preexposed to 0.5% O2 and low glucose concentration for 24 h. These findings indicate that the regulation of HO expression in the human placenta is a complex process that depends, at least in part, on local glucose and oxygen concentrations.

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Abstract TP376: Heme Oxygenase-1 is Essential for Hematoma Clearance After Intracerebral Hemorrhage

Stroke ◽

10.1161/str.48.suppl_1.tp376 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Liyan Zhang ◽

Xiurong Zhao ◽

Guanghua Sun ◽

Jaroslaw Aronowski

Keyword(s):

Intracerebral Hemorrhage ◽

Rat Brain ◽

Heme Oxygenase ◽

Functional Recovery ◽

Protoporphyrin Ix ◽

Neuronal Injury ◽

Neurological Deficits ◽

Sublethal Dose ◽

Heme Oxygenase 1 ◽

Secondary Brain Injury

Background: After intracerebral hemorrhage (ICH), the red blood cells (RBC) and their hemolytic products within brain hematoma trigger adverse biochemical events, leading to secondary brain injury and neurological deficits. Thus, efficient removal of hematoma components is essential for achieving inflammation resolution and functional recovery. The inducible heme-oxygenase (HO-1) is a key rate-limiting enzyme that catabolizes heme into iron, CO, and biliverdin. The present study investigated the role of HO-1 in microglia/macrophages (MΦ)-mediated phagocytosis of RBC; and also assessed the spatial and temporal expression of HO-1 in ICH-affected brain, as well as its possible role in the clearance of hematoma components following ICH modeled in rodents. Methods and Results: First, we employed the rat brain MΦ. Upon exposing to RBC, MΦ phagocytize RBC; and HO-1 was induced during this process. Co-incubating tin-protoporphyrin IX (SnPP, a competitive HO-1 inhibitor) with RBC significantly delayed RBC internalization by MΦ. Removal of SnPP from the culture medium led to a rapid recovery of MΦ’s phagocytic function, suggesting that SnPP-induced inhibition is a reversible process. Subjecting neuron-microglia co-cultures to RBC plus sublethal dose of oxygen-deprivation (an ICH-like insult) triggered neuronal injury, as assessed using neurofilament degradation assay and loss of NeuN-positive cells; and addition of SnPP further aggravated the neuronal injury. Additional studies showed that after ICH, HO-1 is up-regulated in hematoma-affected rat brain tissues starting from 6h, reaching the maximum level at 3-7days, and persisting for at least 10 days after ICH. Double immunohistochemistry of HO-1 and brain cell markers shows that the most HO-1-positive cells are Iba1-positive MΦ. Administration of SnPP for 7 days, (7.5 mg/kg, ip, twice a day) delayed hematoma clearance by 27.8% and significantly impaired the functional recovery, as measured 7 days after ICH. Histological analyses showed that there are more TUNEL-positive neurons in the hematoma-affected brain tissue in SnPP-treated mouse brains. Conclusion: Our study suggests that HO-1 is essential for phagocytosis of RBC by MΦ, which is critical for endogenous clearance of hematoma after ICH.

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Photosystem Disorder Could be the Key Cause for the Formation of Albino Leaf Phenotype in Pecan

International Journal of Molecular Sciences ◽

10.3390/ijms21176137 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6137

Author(s):

Ji-Yu Zhang ◽

Tao Wang ◽

Zhan-Hui Jia ◽

Zhong-Ren Guo ◽

Yong-Zhi Liu ◽

...

Keyword(s):

Chlorophyll A ◽

Molecular Mechanisms ◽

Chlorophyll Biosynthesis ◽

Protoporphyrin Ix ◽

Photosynthetic Electron Transport ◽

Chlorophyll Degradation ◽

Pheophorbide A ◽

Leaf Phenotype ◽

Photosynthesis Pathway ◽

Almost All

Pecan is one of the most famous nut species in the world. The phenotype of mutants with albino leaves was found in the process of seeding pecan, providing ideal material for the study of the molecular mechanisms leading to the chlorina phenotype in plants. Both chlorophyll a and chlorophyll b contents in albino leaves (ALs) were significantly lower than those in green leaves (GLs). A total of 5171 differentially expression genes (DEGs) were identified in the comparison of ALs vs. GLs using high-throughput transcriptome sequencing; 2216 DEGs (42.85%) were upregulated and 2955 DEGs (57.15%) were downregulated. The expressions of genes related to chlorophyll biosynthesis (HEMA1, encoding glutamyl-tRNA reductase; ChlH, encoding Mg-protoporphyrin IX chelatase (Mg-chelatase) H subunit; CRD, encoding Mg-protoporphyrin IX monomethylester cyclase; POR, encoding protochlorophyllide reductase) in ALs were significantly lower than those in GLs. However, the expressions of genes related to chlorophyll degradation (PAO, encoding pheophorbide a oxygenase) in ALs were significantly higher than those in GLs, indicating that disturbance of chlorophyll a biosynthesis and intensification of chlorophyll degradation lead to the absence of chlorophyll in ALs of pecan. A total of 72 DEGs associated with photosynthesis pathway were identified in ALs compared to GLs, including photosystem I (15), photosystem II (19), cytochrome b6-f complex (3), photosynthetic electron transport (6), F-type ATPase (7), and photosynthesis-antenna proteins (22). Moreover, almost all the genes (68) mapped in the photosynthesis pathway showed decreased expression in ALs compared to GLs, declaring that the photosynthetic system embedded within the thylakoid membrane of chloroplast was disturbed in ALs of pecan. This study provides a theoretical basis for elucidating the molecular mechanism underlying the phenotype of chlorina seedlings of pecan.

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