ORCID Profile
0000-0002-4616-2967
Current Organisations
Australian National University
,
University of Newcastle Australia
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Publisher: Springer Science and Business Media LLC
Date: 23-01-2019
DOI: 10.1038/S41586-019-0880-5
Abstract: Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency
Publisher: Oxford University Press (OUP)
Date: 19-03-2010
Abstract: Carboxysomes are an essential part of the cyanobacterial CO2-concentrating mechanism, consisting of a protein shell and an interior of Rubisco. The β-carboxysome shell protein CcmM forms two peptides via a proposed internal ribosomal entry site (IRES) within the ccmM transcript in Synechococcus PCC7942. The abundant short form (35 kD, M35) consists of Rubisco small subunit-like repeats and binds Rubisco. The lower abundance long form (58 kD, M58) also contains a γ-carbonic anhydrase-like domain, which binds the carboxysomal carbonic anhydrase, CcaA. We examined whether these CcmM forms arise via an IRES or by other means. Mutations of a putative internal start codon (GTG) and Shine-Dalgarno sequence within ccmM, along with a gene coding for M35 alone, were examined in the high-CO2-requiring (HCR) carboxysomeless mutant, ΔccmM. Expression of wild-type ccmM in ΔccmM restored the wild-type phenotype, while mutation of putative start and Shine-Dalgarno sequences led to as much as 20-fold reduction in M35 content with no recovery from HCR phenotype. These cells also contained small electron-dense structures. Cells producing little or no M58, but sufficient M35, were found to contain large electron-dense structures, no CcaA, and had a HCR phenotype. Large subcellular aggregates can therefore form in the absence of M58, suggesting a role for M35 in internal carboxysome Rubisco packing. The results confirm that M35 is independently translated via an IRES within ccmM. Importantly, the data reveal that functional carboxysomes require both M35 and M58 in sufficient quantities and with a minimum stoichiometry of close to 1:1.
Publisher: Springer International Publishing
Date: 2021
Publisher: American Society for Microbiology
Date: 2001
DOI: 10.1128/AEM.67.1.278-283.2001
Abstract: Cell quotas of microcystin ( Q MCYST femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a ), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q MCYST and specific growth rate (μ), from which we propose a generalized model that enables Q MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q MCYST from μ, μ max (maximum specific growth rate), Q MCYSTmax (maximum cell quota), and Q MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q MCYSTmax of 0.129 fmol cell −1 at μ max and a Q MCYSTmin of 0.050 fmol cell −1 at μ = 0. Net MCYST production rate ( R MCYST ) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell −1 day −1 at μ max . MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST rotein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.
Publisher: Oxford University Press (OUP)
Date: 19-06-2007
DOI: 10.1093/JXB/ERM112
Abstract: Cyanobacteria have evolved a significant environmental adaptation, known as a CO(2)-concentrating-mechanism (CCM), that vastly improves photosynthetic performance and survival under limiting CO(2) concentrations. The CCM functions to transport and accumulate inorganic carbon actively (Ci HCO(3)(-), and CO(2)) within the cell where the Ci pool is utilized to provide elevated CO(2) concentrations around the primary CO(2)-fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). In cyanobacteria, Rubisco is encapsulated in unique micro-compartments known as carboxysomes. Cyanobacteria can possess up to five distinct transport systems for Ci uptake. Through database analysis of some 33 complete genomic DNA sequences for cyanobacteria it is evident that considerable ersity exists in the composition of transporters employed, although in many species this ersity is yet to be confirmed by comparative phenomics. In addition, two types of carboxysomes are known within the cyanobacteria that have apparently arisen by parallel evolution, and considerable progress has been made towards understanding the proteins responsible for carboxysome assembly and function. Progress has also been made towards identifying the primary signal for the induction of the subset of CCM genes known as CO(2)-responsive genes, and transcriptional regulators CcmR and CmpR have been shown to regulate these genes. Finally, some prospects for introducing cyanobacterial CCM components into higher plants are considered, with the objective of engineering plants that make more efficient use of water and nitrogen.
Publisher: Springer Science and Business Media LLC
Date: 17-04-2023
Publisher: Elsevier BV
Date: 10-2023
Publisher: Springer Science and Business Media LLC
Date: 03-2003
Publisher: Cold Spring Harbor Laboratory
Date: 31-07-2023
DOI: 10.1101/2023.07.31.551272
Abstract: Cyanobacterial CO 2 concentrating mechanisms (CCMs) sequester a globally significant proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO 2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in ɑ-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present the first structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium PCC7001 . Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate (RuBP), and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial ɑ-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. The carboxysomal carbonic anhydrase, CsoSCA, is allosterically activated by the Rubisco substrate RuBP, revealing a novel mechanism controlling key enzyme activity in cyanobacterial α-carboxysomes.
Publisher: Wiley
Date: 03-01-2017
DOI: 10.1111/GCB.13566
Abstract: Understanding of the extent of acclimation of light-saturated net photosynthesis (A
Publisher: Wiley
Date: 20-10-2023
DOI: 10.1111/PCE.14453
Abstract: Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of in idual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state‐of‐the‐art cross‐scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme‐limited ( A c ) and electron transport‐limited ( A j ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear‐by‐location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C 3 wheat and C 4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing A c alone generate more consistent but smaller yield gains across all water and nitrogen environments, A j enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both A c and A j generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross‐scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.
Publisher: Wiley
Date: 08-07-2016
DOI: 10.1111/NPH.14079
Abstract: We examined whether variations in photosynthetic capacity are linked to variations in the environment and/or associated leaf traits for tropical moist forests ( TMF s) in the Andes/western Amazon regions of Peru. We compared photosynthetic capacity (maximal rate of carboxylation of Rubisco ( V cmax ), and the maximum rate of electron transport ( J max )), leaf mass, nitrogen (N) and phosphorus (P) per unit leaf area ( M a , N a and P a , respectively), and chlorophyll from 210 species at 18 field sites along a 3300‐m elevation gradient. Western blots were used to quantify the abundance of the CO 2 ‐fixing enzyme Rubisco. Area‐ and N‐based rates of photosynthetic capacity at 25°C were higher in upland than lowland TMF s, underpinned by greater investment of N in photosynthesis in high‐elevation trees. Soil [P] and leaf P a were key explanatory factors for models of area‐based V cmax and J max but did not account for variations in photosynthetic N‐use efficiency. At any given N a and P a , the fraction of N allocated to photosynthesis was higher in upland than lowland species. For a small subset of lowland TMF trees examined, a substantial fraction of Rubisco was inactive. These results highlight the importance of soil‐ and leaf‐P in defining the photosynthetic capacity of TMF s, with variations in N allocation and Rubisco activation state further influencing photosynthetic rates and N‐use efficiency of these critically important forests.
Publisher: Elsevier BV
Date: 10-2007
Publisher: Canadian Science Publishing
Date: 07-2005
DOI: 10.1139/B05-058
Abstract: Carboxysomes are protein-bound, polyhedral microbodies within cyanobacteria, containing the key enzyme for photosynthetic CO 2 fixation, ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). Sequencing of cyanobacterial genomes has revealed that cyanobacteria possess one or other of two types of carboxysomes. Cyanobacteria containing form 1A Rubisco possess α-carboxysomes, while those with form 1B Rubisco possess β-carboxysomes. Given the central importance of carboxysomes in the CO 2 -concentrating mechanism of cyanobacteria, understanding the nature and composition of these structures is of considerable importance. In an effort to develop techniques for the characterization of the structure of β-carboxysomes, particularly the outer protein shell, we have undertaken a proteomic assessment of the PercollMg 2+ carboxysome enrichment technique using the freshwater cyanobacterium Synechococcus sp. PCC7942. Both matrix-assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF MS) and multidimensional protein identification technology (MuDPIT) methods were used to determine the protein content of a novel carboxysome-rich fraction. A total of 17 proteins were identified using MALDI-TOF MS from enriched carboxysome preparations, while 122 proteins were identified using MuDPIT analysis on the same material. The carboxysomal protein CcmM was identified by MALDI-TOF MS as two distinct proteins of 38 and 58 kDa. The only other carboxysomal proteins identified were the large and small subunits of Rubisco (RbcL and RbcS). Reasons for the lack of evidence for the expected full complement of carboxysomal proteins and future directions are discussed.Key words: CO 2 -concentrating mechanism, cyanobacteria, carboxysomes, proteomics.
Publisher: S. Karger AG
Date: 2013
DOI: 10.1159/000351342
Abstract: Carboxysomes are extraordinarily efficient proteinaceous microcompartments that encapsulate the primary CO sub /sub -fixing enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) in cyanobacteria and some proteobacteria. These microbodies form part of a CO sub /sub -concentrating mechanism b /b (CCM), operating together with active CO sub /sub and HCO sub /sub sup - /sup uptake transporters which accumulate HCO sub /sub sup - /sup in the cytoplasm of the cell. Cyanobacteria (also known as blue-green algae) are highly productive on a global scale, especially those species from open-ocean niches, which collectively contribute nearly 30% of global net primary fixation. This productivity would not be possible without a CCM which is dependent on carboxysomes. Two evolutionarily distinct forms of carboxysome are evident that encapsulate proteobacterial RuBisCO form-1A or higher-plant RuBisCO form- 1B, respectively. Based partly on RuBisCO phylogeny, the two carboxysome types are known either as α-carboxysomes, found in predominantly oceanic cyanobacteria (α-cyanobacteria) and some proteobacteria, or as β-carboxysomes, found mainly in freshwater/estuarine cyanobacteria (β-cyanobacteria). Both carboxysome types are believed to have evolved in parallel as a consequence of fluctuating atmospheric CO sub /sub levels and evolutionary pressure acting via the poor enzymatic kinetics of RuBisCO. The three-dimensional structures and protein components of each carboxysome type reflect distinct evolutionarily strategies to the same major functions: subcellular compartmentalization and RuBisCO encapsulation, oxygen exclusion, and CO sub /sub concentration and fixation.
Publisher: Elsevier BV
Date: 12-2011
Publisher: Cambridge University Press (CUP)
Date: 06-2001
Publisher: Proceedings of the National Academy of Sciences
Date: 30-04-2021
Abstract: Rubisco is arguably the most abundant protein on Earth, and its catalytic action is responsible for the bulk of organic carbon in the biosphere. Its function has been the focus of study for many decades, but recent discoveries highlight that in a broad array of organisms, it undergoes liquid–liquid phase separation to form membraneless organelles, known as pyrenoids and carboxysomes, that enhance CO 2 acquisition. We assess the benefit of these condensate compartments to Rubisco function using a mathematical model. Our model shows that proton production via Rubisco reactions, and those carried by protonated reaction species, can enable the elevation of condensate CO 2 to enhance carboxylation. Application of this theory provides insights into pyrenoid and carboxysome evolution.
Publisher: Springer Science and Business Media LLC
Date: 09-03-2023
DOI: 10.1007/S11120-023-01009-X
Abstract: Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO 2 environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields. To date, two carboxysome types have been characterized, the α-type that has fewer shell components and the β-type that houses a faster Rubisco. While research is underway to construct a native carboxysome in planta , work investigating the internal arrangement of carboxysomes has identified conserved Rubisco amino acid residues between the two carboxysome types which could be engineered to produce a new, hybrid carboxysome. In theory, this hybrid carboxysome would benefit from the simpler α-carboxysome shell architecture while simultaneously exploiting the higher Rubisco turnover rates in β-carboxysomes. Here, we demonstrate in an Escherichia coli expression system, that the Thermosynechococcus elongatus Form IB Rubisco can be imperfectly incorporated into simplified Cyanobium α-carboxysome-like structures. While encapsulation of non-native cargo can be achieved, T. elongatus Form IB Rubisco does not interact with the Cyanobium carbonic anhydrase, a core requirement for proper carboxysome functionality. Together, these results suggest a way forward to hybrid carboxysome formation.
Publisher: Public Library of Science (PLoS)
Date: 22-08-2012
Publisher: American Society for Microbiology
Date: 09-2013
Abstract: Cyanobacteria are the globally dominant photoautotrophic lineage. Their success is dependent on a set of adaptations collectively termed the CO 2 -concentrating mechanism (CCM). The purpose of the CCM is to support effective CO 2 fixation by enhancing the chemical conditions in the vicinity of the primary CO 2 -fixing enzyme, d -ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to promote the carboxylase reaction and suppress the oxygenase reaction. In cyanobacteria and some proteobacteria, this is achieved by encapsulation of RubisCO within carboxysomes, which are ex les of a group of proteinaceous bodies called bacterial microcompartments. Carboxysomes encapsulate the CO 2 -fixing enzyme within the selectively permeable protein shell and simultaneously encapsulate a carbonic anhydrase enzyme for CO 2 supply from a cytoplasmic bicarbonate pool. These bodies appear to have arisen twice and undergone a process of convergent evolution. While the gross structures of all known carboxysomes are ostensibly very similar, with shared gross features such as a selectively permeable shell layer, each type of carboxysome encapsulates a phyletically distinct form of RubisCO enzyme. Furthermore, the specific proteins forming structures such as the protein shell or the inner RubisCO matrix are not identical between carboxysome types. Each type has evolutionarily distinct forms of the same proteins, as well as proteins that are entirely unrelated to one another. In light of recent developments in the study of carboxysome structure and function, we present this review to summarize the knowledge of the structure and function of both types of carboxysome. We also endeavor to cast light on differing evolutionary trajectories which may have led to the differences observed in extant carboxysomes.
Publisher: Frontiers Media SA
Date: 31-08-2021
Abstract: Heterologous synthesis of a biophysical CO 2 -concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C 3 plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO 2 concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C 3 plant chloroplast is functional bicarbonate (HCO 3 − ) transporters and vectorial CO 2 -to-HCO 3 − converters. Engineering strategies aim to locate these transporters and conversion systems to the C 3 chloroplast, enabling elevation of HCO 3 − concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C 3 plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO 3 − transporter, and CO 2 -to-HCO 3 − converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes.
Publisher: Springer Science and Business Media LLC
Date: 21-11-2019
Publisher: Cold Spring Harbor Laboratory
Date: 10-07-2020
DOI: 10.1101/2020.07.08.125609
Abstract: Membraneless organelles containing the enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO 2 concentrating mechanisms (CCMs) to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO 2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO 2 within the organelle via a co-localized carbonic anhydrase (CA), and inwardly diffusing HCO 3 - which has accumulated in the cytoplasm via dedicated transporters. Here we present a novel concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction-diffusion, compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3 - accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO 3 - to CO 2 via co-localized CA, enhancing both condensate [CO 2 ] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (PGA) plays an important role in modulating internal pH and CO 2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO 3 - accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully-formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO 2 and low light environments.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.PBI.2016.03.002
Abstract: Global population growth is projected to outpace plant-breeding improvements in major crop yields within decades. To ensure future food security, multiple creative efforts seek to overcome limitations to crop yield. Perhaps the greatest limitation to increased crop yield is photosynthetic inefficiency, particularly in C3 crop plants. Recently, great strides have been made toward crop improvement by researchers seeking to introduce the cyanobacterial CO2-concentrating mechanism (CCM) into plant chloroplasts. This strategy recognises the C3 chloroplast as lacking a CCM, and being a primordial cyanobacterium at its essence. Hence the collection of solute transporters, enzymes, and physical structures that make cyanobacterial CO2-fixation so efficient are viewed as a natural source of genetic material for C3 chloroplast improvement. Also we highlight recent outstanding research aimed toward the goal of introducing a cyanobacterial CCM into C3 chloroplasts and consider future research directions.
Publisher: Oxford University Press (OUP)
Date: 10-10-2005
DOI: 10.1093/JXB/ERI286
Abstract: Cyanobacteria probably exhibit the widest range of ersity in growth habitats of all photosynthetic organisms. They are found in cold and hot, alkaline and acidic, marine, freshwater, saline, terrestrial, and symbiotic environments. In addition to this, they originated on earth at least 2.5 billion years ago and have evolved through periods of dramatic O2 increases, CO2 declines, and temperature changes. One of the key problems they have faced through evolution and in their current environments is the capture of CO2 and its efficient use by Rubisco in photosynthesis. A central response to this challenge has been the development of a CO2 concentrating mechanism (CCM) that can be adapted to various environmental limitations. There are two primary functional elements of this CCM. Firstly, the containment of Rubisco in carboxysome protein microbodies within the cell (the sites of CO2) elevation), and, secondly, the presence of several inorganic carbon (Ci) transporters that deliver HCO3- intracellularly. Cyanobacteria show both species adaptation and acclimation of this mechanism. Between species, there are differences in the suites of Ci transporters in each genome, the nature of the carboxysome structures and the functional roles of carbonic anhydrases. Within a species, different CCM activities can be induced depending on the Ci availability in the environment. This acclimation is largely based on the induction of multiple Ci transporters with different affinities and specificities for either CO2 or HCO3- as substrates. These features are discussed in relation to our current knowledge of the genomic sequences of a erse array of cyanobacteria and their ecological environments.
Publisher: Wiley
Date: 12-05-2015
DOI: 10.1111/PCE.12544
Abstract: In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of in idual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.
Publisher: Springer Science and Business Media LLC
Date: 08-06-2014
DOI: 10.1007/S11120-014-0018-4
Abstract: Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2011
DOI: 10.1007/S11120-011-9659-8
Abstract: Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.
Publisher: Proceedings of the National Academy of Sciences
Date: 27-10-2023
Publisher: Springer Science and Business Media LLC
Date: 03-09-2018
DOI: 10.1038/S41467-018-06044-0
Abstract: A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO 2 -concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO 2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO 2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium , within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO 2 . This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.
Publisher: Oxford University Press (OUP)
Date: 18-03-2014
Publisher: Oxford University Press (OUP)
Date: 24-04-2017
DOI: 10.1093/JXB/ERX133
Abstract: Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.
Publisher: Elsevier BV
Date: 2010
Publisher: Oxford University Press (OUP)
Date: 11-2017
DOI: 10.1093/JXB/ERX351
Publisher: American Chemical Society (ACS)
Date: 19-10-2021
Publisher: Elsevier BV
Date: 10-2000
DOI: 10.1016/S1095-6433(00)00247-6
Abstract: The concentrations of free amino acids were measured in whole claw muscle, single fibres and haemolymph of Australian freshwater crayfish, Cherax destructor, during the intermoult stage. The average total pool of amino acids in short-sarcomere fibres (179 mmol kg(-1)) was 60% greater than in long-sarcomere fibres, due to higher concentrations of alanine, cysteine, glutamate, leucine and proline. The two fibre types exhibited differences in the banding pattern of the isoforms of troponin using gel electrophoresis. The average pool of amino acids in haemolymph was 2.7 mmol kg(-1). Cherax has symmetrical claws and the total pool of amino acids from whole muscles (approx. 79 mmol kg(-1)) was similar in left and right claw muscles. In animals acclimated to osmotic environments between 0 and 220 mOsm, the osmotic pressure of the haemolymph increased from 356 to 496 mOsm, but no systematic changes were observed in the amino acid profiles of muscles or haemolymph. The major findings were that (a) concentrations of amino acids differed between the two major fibre types in claw muscle and (b) amino acids in the muscle fibres did not play a major part in intracellular osmoregulation in Cherax, suggesting this species is an anisosmotic regulator.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Benedict Long.