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

1999 ◽  
Vol 337 (2) ◽  
pp. 243-251 ◽  
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.

scholarly journals Inactivation of Mg Chelatase during Transition from Anaerobic to Aerobic Growth in Rhodobacter capsulatus

2003 ◽  
Vol 185 (11) ◽  
pp. 3249-3258 ◽  
Author(s):  
Robert D. Willows ◽  
Vanessa Lake ◽  
Thomas Hugh Roberts ◽  
Samuel I. Beale
Keyword(s):  
Rhodobacter Capsulatus ◽  
Control Point ◽  
Protoporphyrin Ix ◽  
Photosynthetic Bacterium ◽  
Aerobic Conditions ◽  
Heme Synthesis ◽  
Cell Extracts ◽  
Heterotrophic Metabolism ◽  
Mg Chelatase

ABSTRACT The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg2+ to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O2, and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O2-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.

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

2019 ◽  
Vol 476 (13) ◽  
pp. 1875-1887 ◽  
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.

scholarly journals ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

2006 ◽  
Vol 400 (3) ◽  
pp. 477-484 ◽  
Author(s):  
Nick Sirijovski ◽  
Ulf Olsson ◽  
Joakim Lundqvist ◽  
Salam Al-Karadaghi ◽  
Robert D. Willows ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Mass Fraction ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
Gel Filtration ◽  
Protoporphyrin Ix ◽  
High Molecular Mass ◽  
Magnesium Chelatase ◽  
Ring Complex

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.

scholarly journals Bilin-dependent regulation of chlorophyll biosynthesis by GUN4

2021 ◽  
Vol 118 (20) ◽  
pp. e2104443118
Author(s):  
Weiqing Zhang ◽  
Robert D. Willows ◽  
Rui Deng ◽  
Zheng Li ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Heme Oxygenase ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Common Trunk ◽  
Magnesium Chelatase ◽  
Photosynthetic Eukaryotes ◽  
Linear Tetrapyrroles ◽  
Oxygenic Phototrophs ◽  
Binding Component

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.

scholarly journals Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54

2020 ◽  
Vol 477 (12) ◽  
pp. 2313-2325 ◽  
Author(s):  
Guangyu E. Chen ◽  
C. Neil Hunter
Keyword(s):  
Protoporphyrin Ix ◽  
Photosynthetic Bacterium ◽  
Normal Function ◽  
Green Algal ◽  
Auxiliary Subunit ◽  
Absolute Requirement ◽  
Model Plant Arabidopsis Thaliana ◽  
Rubrivivax Gelatinosus ◽  
Oxygenic Phototrophs

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit.

1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis

2017 ◽  
Vol 474 (12) ◽  
pp. 2095-2105 ◽  
Author(s):  
Artur Sawicki ◽  
Shuaixiang Zhou ◽  
Kathrin Kwiatkowski ◽  
Meizhong Luo ◽  
Robert D. Willows
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Magnesium Chelatase ◽  
Orthologous Protein ◽  
Substrate Complex ◽  
Motor Complex ◽  
Histidine Phosphorylation ◽  
Mg Chelatase ◽  
Stimulation Of

Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme ‘motor’ complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.

scholarly journals Structure of GUN4 fromChlamydomonas reinhardtii

2015 ◽  
Vol 71 (8) ◽  
pp. 1094-1099 ◽  
Author(s):  
Shabnam Tarahi Tabrizi ◽  
David B. Langley ◽  
Stephen J. Harrop ◽  
Anthony P. Duff ◽  
Robert D. Willows
Keyword(s):  
Crystal Structure ◽  
Chlamydomonas Reinhardtii ◽  
Chlorophyll Biosynthesis ◽  
Conformational Dynamics ◽  
Protoporphyrin Ix ◽  
Biosynthesis Pathway ◽  
Broad Agreement ◽  
Binding Cleft ◽  
Mg Chelatase

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.

scholarly journals MUCUS IN INTESTINAL CONTENTS OF GERMFREE RATS

1965 ◽  
Vol 121 (2) ◽  
pp. 201-213 ◽  
Author(s):  
Göran Lindstedt ◽  
Sven Lindstedt ◽  
Bengt E. Gustafsson
Keyword(s):  
Gel Filtration ◽  
Intestinal Flora ◽  
Test System ◽  
Nitrogen Excretion ◽  
Fecal Excretion ◽  
Cecal Wall ◽  
Highly Active ◽  
Germfree Rats

The fecal excretion of total nitrogen and of total hexosamines has been determined in germfree and conventional rats. Germfree rats excreted more hexosamines than the conventional rats, while no difference in the nitrogen excretion was found. Infection of the germfree rats with a normal flora resulted in a temporarily increased excretion of hexosamines and nitrogen over a period of 2 to 3 days after which they reached the level of the conventional animals. The contents of the germfree cecum contained 65 to 137 mg of hexosamines and 57 to 127 mg of nitrogen as compared to 1.2 to 5.3 and 7.4 to 23 mg in conventional animals. The high figures for hexosamines were due to an increase in the total amount of contents in the cecum and to a fivefold increase in the concentration of hexosamine-containing material. Studies on the distribution of hexosamine-containing cecal contents between sediment and supernatant after centrifugation at 20,000 g for 2 hours demonstrated that 5 to 10 per cent of the hexosamines occurred in the sediment in the germfree rats, while 75 to 85 per cent was found in this fraction in the conventional rats. The soluble part of the cecal contents in germfree as well as in the conventional rats contained 70 per cent of hexosamines in molecules with a molecular weight above approximatively 100,000 as found by gel filtration experiments on sephadex gels. The higher weight of the germfree cecal wall was reflected in a high total amount of nitrogen and hexosamines. Isolated strains of bacteria capable of reducing the cecal size in vivo did not show any capacity to degrade the mucus in vitro in a test system, where a full intestinal flora was highly active.

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

2000 ◽  
Vol 352 (2) ◽  
pp. 435-441 ◽  
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.

Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12

1993 ◽  
Vol 39 (12) ◽  
pp. 1155-1161 ◽  
Author(s):  
Alexandre Sasarman ◽  
Jaroslav Letowski ◽  
Guy Czaika ◽  
Volta Ramirez ◽  
Michael A. Nead ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Oxidase Activity ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Translation System ◽  
Protoporphyrinogen Oxidase ◽  
Escherichia Coli K12 ◽  
Amino Terminal

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21 202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription–translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.Key words: protoporphyrinogen oxidase (PROTOX), hemG gene, Escherichia coli, DPE herbicides, heme.

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