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13 C flux analysis of cyanobacterial metabolism.
Adebiyi AO, Jazmin LJ, Young JD
(2015) Photosynth Res 126: 19-32
MeSH Terms: Carbon Isotopes, Cyanobacteria, Metabolic Flux Analysis, Software
Show Abstract · Added January 23, 2015
(13)C metabolic flux analysis (MFA) has made important contributions to our understanding of the physiology of model strains of E. coli and yeast, and it has been widely used to guide metabolic engineering efforts in these microorganisms. Recent advancements in (13)C MFA methodology combined with publicly available software tools are creating new opportunities to extend this approach to examine less characterized microbes. In particular, growing interest in the use of cyanobacteria as industrial hosts for photosynthetic production of biofuels and biochemicals has led to a critical need to better understand how cyanobacterial metabolic fluxes are regulated in response to changes in growth conditions or introduction of heterologous pathways. In this contribution, we review several prior studies that have applied isotopic steady-state (13)C MFA to examine heterotrophic or mixotrophic growth of cyanobacteria, as well as recent studies that have pioneered the use of isotopically nonstationary MFA (INST-MFA) to study autotrophic cultures. We also provide recommendations for the design and analysis of INST-MFA experiments in cyanobacteria, based on our previous experience and a series of simulation studies used to assess the selection of measurements and sample time points. We anticipate that this emerging knowledgebase of prior (13)C MFA studies, optimized experimental protocols, and public software tools will catalyze increasing use of (13)C MFA techniques by the cyanobacteria research community.
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4 MeSH Terms
Intricate protein-protein interactions in the cyanobacterial circadian clock.
Egli M
(2014) J Biol Chem 289: 21267-75
MeSH Terms: Amino Acid Sequence, Bacterial Proteins, Circadian Clocks, Cyanobacteria, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Conformation
Show Abstract · Added June 26, 2014
The cyanobacterial circadian clock consists of a post-translational oscillator (PTO) and a PTO-dependent transcription-translation feedback loop (TTFL). The PTO can be reconstituted in vitro with the KaiA, KaiB, and KaiC proteins, enabling detailed biochemical and biophysical investigations. Both the CI and the CII halves of the KaiC hexamer harbor ATPases, but only the C-terminal CII ring exhibits kinase and phospho-transferase activities. KaiA stimulates the kinase and KaiB associates with KaiC during the dephosphorylation phase and sequesters KaiA. Recent research has led to conflicting models of the KaiB-KaiC interaction, precluding a clear understanding of KaiB function and KaiABC clock mechanism.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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8 MeSH Terms
Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.
Johnson CH, Egli M
(2014) Annu Rev Biochem 83: 221-47
MeSH Terms: Bacterial Proteins, Circadian Clocks, Circadian Rhythm, Circadian Rhythm Signaling Peptides and Proteins, Cyanobacteria, Feedback, Physiological, Gene Expression Regulation, Bacterial, Homeostasis, Protein Biosynthesis, Protein Processing, Post-Translational, Temperature, Transcription, Genetic
Show Abstract · Added June 26, 2014
For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value.
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1 Members
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12 MeSH Terms
A circadian clock nanomachine that runs without transcription or translation.
Egli M, Johnson CH
(2013) Curr Opin Neurobiol 23: 732-40
MeSH Terms: Animals, Bacterial Physiological Phenomena, Bacterial Proteins, Circadian Clocks, Circadian Rhythm, Cyanobacteria, Nanomedicine
Show Abstract · Added March 7, 2014
The biochemical basis of circadian timekeeping is best characterized in cyanobacteria. The structures of its key molecular players, KaiA, KaiB, and KaiC are known and these proteins can reconstitute a remarkable circadian oscillation in a test tube. KaiC is rhythmically phosphorylated and its phospho-status is a marker of circadian phase that regulates ATPase activity and the oscillating assembly of a nanomachine. Analyses of the nanomachines have revealed how their timing circuit is ratcheted to be unidirectional and how they stay in synch to ensure a robust oscillator. These insights are likely to elucidate circadian timekeeping in higher organisms, including how transcription and translation could appear to be a core circadian timer when the true pacemaker is an embedded biochemical oscillator.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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7 MeSH Terms
Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH
(2012) Biochemistry 51: 1547-58
MeSH Terms: Adenosine Triphosphatases, Amino Acid Sequence, Bacterial Proteins, CLOCK Proteins, Catalytic Domain, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Cyanobacteria, Models, Molecular, Molecular Sequence Data, Mutation, Phosphorylation
Show Abstract · Added March 7, 2014
The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.
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12 MeSH Terms
The cyanobacterial circadian system: from biophysics to bioevolution.
Johnson CH, Stewart PL, Egli M
(2011) Annu Rev Biophys 40: 143-67
MeSH Terms: Bacterial Proteins, Biological Clocks, Circadian Rhythm, Computer Simulation, Cyanobacteria, Evolution, Molecular, Models, Biological
Show Abstract · Added March 7, 2014
Recent studies have unveiled the molecular machinery responsible for the biological clock in cyanobacteria and found that it exerts pervasive control over cellular processes including global gene expression. Indeed, the entire chromosome undergoes daily cycles of topology/compaction! The circadian system comprises both a posttranslational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO can be reconstituted in vitro with three purified proteins (KaiA, KaiB, and KaiC) and ATP. These are the only circadian proteins for which high-resolution structures are available. Phase in this nanoclockwork has been associated with key phosphorylations of KaiC. Structural considerations illuminate the mechanism by which the KaiABC oscillator ratchets unidirectionally. Models of the complete in vivo system have important implications for our understanding of circadian clocks in higher organisms, including mammals. The conjunction of structural, biophysical, and biochemical approaches to this system has brought our understanding of the molecular mechanisms of biological timekeeping to an unprecedented level.
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2 Members
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7 MeSH Terms
Circadian clocks and cell division: what's the pacemaker?
Johnson CH
(2010) Cell Cycle 9: 3864-73
MeSH Terms: Animals, Biological Evolution, Cell Cycle, Circadian Clocks, Circadian Rhythm, Circadian Rhythm Signaling Peptides and Proteins, Cyanobacteria, Euglena, Feedback, Physiological, Humans, Light
Show Abstract · Added February 12, 2015
Evolution has selected a system of two intertwined cell cycles: the cell division cycle (CDC) and the daily (circadian) biological clock. The circadian clock keeps track of solar time and programs biological processes to occur at environmentally appropriate times. One of these processes is the CDC, which is often gated by the circadian clock. The intermeshing of these two cell cycles is probably responsible for the observation that disruption of the circadian system enhances susceptibility to some kinds of cancer. The core mechanism underlying the circadian clockwork has been thought to be a transcription & translation feedback loop (TTFL), but recent evidence from studies with cyanobacteria, synthetic oscillators and immortalized cell lines suggests that the core circadian pacemaking mechanism that gates cell division in mammalian cells could be a post-translational oscillator (PTO).
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11 MeSH Terms
Intramolecular regulation of phosphorylation status of the circadian clock protein KaiC.
Xu Y, Mori T, Qin X, Yan H, Egli M, Johnson CH
(2009) PLoS One 4: e7509
MeSH Terms: Bacterial Proteins, Binding Sites, Circadian Rhythm, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Cyanobacteria, Gene Expression Regulation, Bacterial, Kinetics, Models, Biological, Mutation, Phosphorylation, Synechococcus
Show Abstract · Added May 27, 2014
BACKGROUND - KaiC, a central clock protein in cyanobacteria, undergoes circadian oscillations between hypophosphorylated and hyperphosphorylated forms in vivo and in vitro. Structural analyses of KaiC crystals have identified threonine and serine residues in KaiC at three residues (T426, S431, and T432) as potential sites at which KaiC is phosphorylated; mutation of any of these three sites to alanine abolishes rhythmicity, revealing an essential clock role for each residue separately and for KaiC phosphorylation in general. Mass spectrometry studies confirmed that the S431 and T432 residues are key phosphorylation sites, however, the role of the threonine residue at position 426 was not clear from the mass spectrometry measurements.
METHODOLOGY AND PRINCIPAL FINDINGS - Mutational approaches and biochemical analyses of KaiC support a key role for T426 in control of the KaiC phosphorylation status in vivo and in vitro and demonstrates that alternative amino acids at residue 426 dramatically affect KaiC's properties in vivo and in vitro, especially genetic dominance/recessive relationships, KaiC dephosphorylation, and the formation of complexes of KaiC with KaiA and KaiB. These mutations alter key circadian properties, including period, amplitude, robustness, and temperature compensation. Crystallographic analyses indicate that the T426 site is phosphorylatible under some conditions, and in vitro phosphorylation assays of KaiC demonstrate labile phosphorylation of KaiC when the primary S431 and T432 sites are blocked.
CONCLUSIONS AND SIGNIFICANCE - T426 is a crucial site that regulates KaiC phosphorylation status in vivo and in vitro and these studies underscore the importance of KaiC phosphorylation status in the essential cyanobacterial circadian functions. The regulatory roles of these phosphorylation sites--including T426--within KaiC enhance our understanding of the molecular mechanism underlying circadian rhythm generation in cyanobacteria.
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12 MeSH Terms
Omega-3 fatty acids are oxygenated at the n-7 carbon by the lipoxygenase domain of a fusion protein in the cyanobacterium Acaryochloris marina.
Gao B, Boeglin WE, Brash AR
(2010) Biochim Biophys Acta 1801: 58-63
MeSH Terms: Bacterial Proteins, Cyanobacteria, Fatty Acids, Omega-3, Kinetics, Lipoxygenase, Protein Structure, Tertiary, Recombinant Fusion Proteins, Substrate Specificity
Show Abstract · Added December 10, 2013
Lipoxygenases (LOX) are found in most organisms that contain polyunsaturated fatty acids, usually existing as individual genes although occasionally encoded as a fusion protein with a catalase-related hemoprotein. Such a fusion protein occurs in the cyanobacterium Acaryochloris marina and herein we report the novel catalytic activity of its LOX domain. The full-length protein and the C-terminal LOX domain were expressed in Escherichia coli, and the catalytic activities characterized by UV, HPLC, GC-MS, and CD. All omega-3 polyunsaturates were oxygenated by the LOX domain at the n-7 position and with R stereospecificity: alpha-linolenic and the most abundant fatty acid in A. marina, stearidonic acid (C18.4omega3), are converted to the corresponding 12R-hydroperoxides, eicosapentaenoic acid to its 14R-hydroperoxide, and docosahexaenoic acid to its 16R-hydroperoxide. Omega-6 polyunsaturates were oxygenated at the n-10 position, forming 9R-hydroperoxy-octadecadienoic acid from linoleic acid and 11R-hydroperoxy-eicosatetraenoic acid from arachidonic acid. The metabolic transformation of stearidonic acid by the full-length fusion protein entails its 12R oxygenation with subsequent conversion by the catalase-related domain to a novel allene epoxide, a likely precursor of cyclopentenone fatty acids or other signaling molecules (Gao et al, J. Biol. Chem. 284:22087-98, 2009). Although omega-3 fatty acids and lipoxygenases are of widespread occurrence, this appears to be the first description of a LOX-catalyzed oxygenation that specifically utilizes the terminal pentadiene of omega-3 fatty acids.
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8 MeSH Terms
Evidence for an ionic intermediate in the transformation of fatty acid hydroperoxide by a catalase-related allene oxide synthase from the Cyanobacterium Acaryochloris marina.
Gao B, Boeglin WE, Zheng Y, Schneider C, Brash AR
(2009) J Biol Chem 284: 22087-98
MeSH Terms: Catalase, Catalysis, Chromatography, Gas, Cloning, Molecular, Cyanobacteria, Fatty Acids, Gene Expression Regulation, Bacterial, Intramolecular Oxidoreductases, Lipid Peroxides, Magnetic Resonance Spectroscopy, Mass Spectrometry, Models, Biological, Models, Chemical, Oxygen, Solvents
Show Abstract · Added December 10, 2013
Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.
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15 MeSH Terms