Dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase in <i>Gloeothece</i>

Du, Caigan; Reade, John P.H.; Rogers, Lyndon J.; Gallon, John

doi:10.1042/bst022332s

Cited by 4 publications

(3 citation statements)

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“…In recent years, there have been increasing efforts directed toward generating proteome-wide maps of PPIs . The most commonly used high-throughput methods for the study of protein complexes are yeast-two-hybrid (Y2H) screens , or affinity purification-mass spectrometry (AP-MS). − The Y2H screens are expensive, time consuming, and incomplete . The N- or C-terminal tagging in the AP-MS method can affect the expression and interaction of endogenous proteins, , and the application of an AP-MS method is also limited by the availability of tagged constructs or antibodies.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

et al. 2018

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The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N fixation (BNF), a process highly sensitive to oxygen. Previous work has focused on determining protein expression levels under different growth conditions. A major gap of our knowledge is an understanding on how these expressed proteins are assembled into complexes and organized into metabolic pathways, an area that has not been thoroughly investigated. Here, we combined size-exclusion chromatography (SEC) with label-free quantitative mass spectrometry (MS) and bioinformatics to characterize many protein complexes from Cyanothece 51142 cells grown under a 12 h light-dark cycle. We identified 1386 proteins in duplicate biological replicates, and 64% of those proteins were identified as putative complexes. Pairwise computational prediction of protein-protein interaction (PPI) identified 74 822 putative interactions, of which 2337 interactions were highly correlated with published protein coexpressions. Many sequential glycolytic and TCA cycle enzymes were identified as putative complexes. We also identified many membrane complexes that contain cytoplasmic domains. Subunits of NDH-1 complex eluted in a fraction with an approximate mass of ∼669 kDa, and subunits composition revealed coexistence of distinct forms of NDH-1 complex subunits responsible for respiration, electron flow, and CO uptake. The complex form of the phycocyanin beta subunit was nonphosphorylated, and the monomer form was phosphorylated at Ser20, suggesting phosphorylation-dependent deoligomerization of the phycocyanin beta subunit. This study provides an analytical platform for future studies to reveal how these complexes assemble and disassemble as a function of diurnal and circadian rhythms.

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Section: Introductionmentioning

confidence: 99%

“…22−24 The Y2H screens are expensive, time consuming, and incomplete. 25 The N-or C-terminal tagging in the AP-MS method can affect the expression and interaction of endogenous proteins, 24,26 and the application of an AP-MS method is also limited by the availability of tagged constructs or antibodies.…”

Section: ■ Introductionmentioning

confidence: 99%

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

et al. 2018

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“…The two forms of the Feprotein that CM be detected in extracts of R rubrum behave very similarly during SDSPAGE to the Fe-protein in extracts of Attempts to demonstrate ADP-nbosylabon of the Fe-protein in Gloeothece have proved unsuccessful For example, incubabon of Gloeothece w~th NaH,'2P04 for 6 h or w~th [8-l4CC] adenine for 24 h faded to generate any radioactwe Fe-protein In addibon, anfibodes rased agrunst ADP-ribose d d not react wth Fe-protein from Gloeothece, though they d d react w~th Fe-protein from R rubrum (J Cheng, H Hilz & J R Gallon, unpublished findings) On the other hand, Gloeothece contam proteins that react wth mfisera rased agmnst the enzymes that catalyse the reversible ADP-nbosylabon of the Fe-protein of R rubrum nitrogenase [6] However, there is no evldence that these Gloeothece proteins catalyse any ADP-nbosylabon reacbon Moreover, even if they do, their substrate is not, apparently, the Fe-protein of nitrogenase…”

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confidence: 99%

MODIFICATION OF THE IRON PROTEIN OF GLOEOTHECE NITROGENASE: A MASS SPECTROMETRIC STUDY

Dougherty

Brown

Gallon

et al. 1996

Biochemical Society Transactions

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Gloeothece is a non-heterocystous, unicellular cyanobacterium that is capable of aerobic N, fixation [I]. As in other diazotrophs, nitrogenase in Gloeothece consists of a MoFe-protein and an Feprotein. The Faprotein of Gloeothece nitrogenase can be resolved into two different, though antigenically similar, forms by SDS-PAGE. These two forms have apparent M, values of 38 500 and 40 000 respectively, and their relative concentrations vary according to prevailing environmental conditions [2].In purple photosynthetic bacteria such as Rhodospirillurn rubrum, the Fe-protein of nitrogenase undergoes reversible covalent modification by ADP-ribosylation [3]. The two forms of the Feprotein that CM be detected in extracts of R rubrum behave very similarly during SDSPAGE to the Fe-protein in extracts of Gloeothece. By analogy, therefore, it is assumed that the larger (M, = 40 000) form of the Gloeothece Faprotein arises by modification of the smaller (M, = 38 500) protein [2,4]. Consistent with this, the relative concentrations of the two forms of the Fe-protein can change even when nitrogenase synthesis is inhibited [2]. However, the nature of the modification of the Fe-protein in Gloeothece and other cyanobacteria [5] is currently unknown.Attempts to demonstrate ADP-nbosylabon of the Fe-protein in Gloeothece have proved unsuccessful For example, incubabon of Gloeothece w~th NaH,'2P04 for 6 h or w~th [8-l4CC] adenine for 24 h faded to generate any radioactwe Fe-protein In addibon, anfibodes rased agrunst ADP-ribose d d not react wth Fe-protein from Gloeothece, though they d d react w~th Fe-protein from R rubrum (J Cheng, H Hilz & J R Gallon, unpublished findings) On the other hand, Gloeothece contam proteins that react wth mfisera rased agmnst the enzymes that catalyse the reversible ADP-nbosylabon of the Fe-protein of R rubrum nitrogenase [6] However, there is no evldence that these Gloeothece proteins catalyse any ADP-nbosylabon reacbon Moreover, even if they do, their substrate is not, apparently, the Fe-protein of nitrogenaseIn an attempt to determine the nature of the modification of the Fe-protein in Gloeothece, both forms of the this protein were punfied by immunoprecipitahon, using antibodies raised against the Fe-protein from R rubrum, and SDSPAGE. The purified proteins were then subjected to digestion with trypsin and the peptide fragments obtained in this way were analyzed by matrix-assisted laser desorptiodionization time of flight (MALDI-TOF) mass spectrometry (MS). Preliminary MALDI-TOF MS of tryptic digests yielded similar spectra for both the larger (presumed modified) and smaller (presumed unmodified) forms of the Fe-protein of Gloeothece nitrogenase (Fig I). This indcates that both forms of the Fe-protein are derivatives of a single polypeptide. More detailed analysis [7,8] will, hopefully, pinpoint differences in the mass spectra of the two forms of the Fe-protein that will, in turn, elucidate both the nature and the precise site of modification of this protein in Gloeothece. Figw 1. MALDbTOF maw s p c b ...

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Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152

et al. 1999

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Dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase in Gloeothece

Cited by 4 publications

References 2 publications

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

MODIFICATION OF THE IRON PROTEIN OF GLOEOTHECE NITROGENASE: A MASS SPECTROMETRIC STUDY

Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152

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