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 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 RegA Control of Bacteriochlorophyll and Carotenoid Synthesis in Rhodobacter capsulatus

2007 ◽  
Vol 189 (21) ◽  
pp. 7765-7773 ◽  
Author(s):  
Jonathan Willett ◽  
James L. Smart ◽  
Carl E. Bauer
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Carotenoid Biosynthesis ◽  
Divergent Promoters ◽  
Anaerobic Induction ◽  
Single Region ◽  
Phytoene Dehydrogenase ◽  
Carotenoid Synthesis

ABSTRACT We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. β-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.

Nitrogenase inactivation by oxygen and enzyme turnover in Anabaena cylindrica

1983 ◽  
Vol 29 (10) ◽  
pp. 1286-1294 ◽  
Author(s):  
Marcia A. Murry ◽  
Patrick C. Hallenbeck ◽  
Diane Esteva ◽  
John R. Benemann
Keyword(s):  
Time Course ◽  
Acetylene Reduction Activity ◽  
Anabaena Cylindrica ◽  
Reactive Material ◽  
Chloromethyl Ketone ◽  
Aerobic Conditions ◽  
Reduction Activity ◽  
Cell Extracts

Nitrogenase is known to be irreversibly inactivated by oxygen in vivo and in vitro. In time-course experiments using Anabaena cylindrica, cultures treated with antibiotics and incubated under various O2 tensions, in vivo acetylene reduction activity and immunologically determined Fe–Mo protein (component I) were lost at rates directly related to O2 tension. Activity was lost at a faster rate than cross-reactive material. The half-life of cross-reactive material was 25 h under microaerophilic conditions, 12 h under aerobic conditions, and 6.6 h under an O2 tension of 245% of air saturation. In vitro, cross-reactive material was lost in O2 exposed, but not in anaerobically prepared, crude cell extracts. Loss of cross-reactive material was prevented by freezing and by α-N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of histidine-residue proteases. These results indicate that nitrogenase is continuously inactivated by O2, hydrolyzed, and resynthesized during growth of this heterocystous cyanobacteria under aerobic conditions.

scholarly journals Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

2000 ◽  
Vol 182 (10) ◽  
pp. 2831-2837 ◽  
Author(s):  
Sylvie Elsen ◽  
Wanda Dischert ◽  
Annette Colbeau ◽  
Carl E. Bauer
Keyword(s):  
Photosynthetic Bacteria ◽  
Rhodobacter Capsulatus ◽  
Regulatory System ◽  
Photosynthetic Bacterium ◽  
Uptake Hydrogenase ◽  
Carbon And Nitrogen ◽  
Cellular Energy ◽  
Purple Photosynthetic Bacteria ◽  
Two Component

ABSTRACT Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but notnifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required fornifA2 expression, RegA acts as a coactivator ofnifA2. We also demonstrated that in regmutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H2 oxidation, as well as the energy-demanding processes of N2 fixation and CO2 assimilation.

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.

Short-Term Regulation of Nitrogenase Activity by NH4+ in Rhodobacter capsulatus: Multiple In Vivo Nitrogenase Responses to NH4+ Addition

1998 ◽  
Vol 180 (23) ◽  
pp. 6392-6395 ◽  
Author(s):  
Alexander F. Yakunin ◽  
Patrick C. Hallenbeck
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Magnitude Response ◽  
Short Term ◽  
Reversible Inhibition

ABSTRACT The photosynthetic bacterium Rhodobacter capsulatus has been shown to carry out nitrogenase “switch-off,” a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and adraT draG mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a “magnitude” response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH4 +.

scholarly journals Interaction between the H2Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

2003 ◽  
Vol 185 (24) ◽  
pp. 7111-7119 ◽  
Author(s):  
Sylvie Elsen ◽  
Ophélie Duché ◽  
Annette Colbeau
Keyword(s):  
Histidine Kinase ◽  
Rhodobacter Capsulatus ◽  
Expression System ◽  
Photosynthetic Bacterium ◽  
Homologous Expression ◽  
Uptake Activity ◽  
Mutant Phenotypes ◽  
In Vitro Interaction

ABSTRACT The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H2 through a signal transduction cascade comprising three proteins: the H2-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His6)-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His6-HupUHupV complex present in dimeric and tetrameric forms, both of which had H2 uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H2 affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H2 regulation.

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.

scholarly journals Enhanced Nitrogenase Activity in Strains of Rhodobacter capsulatus That Overexpress the rnf Genes

2000 ◽  
Vol 182 (5) ◽  
pp. 1208-1214 ◽  
Author(s):  
Ho-Sang Jeong ◽  
Yves Jouanneau
Keyword(s):  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Wild Type Level ◽  
Intracellular Content ◽  
Nitrogenase Activities ◽  
Membrane Bound

ABSTRACT In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEHoperon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex inR. capsulatus. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex.

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