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1.
PLoS One ; 15(3): e0229897, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32142525

RESUMO

Physiology and nutritional quality of a facultative CAM plant Mesembryanthemum crystallinum under drought stress alone are poorly understood. To induce drought, M. crystallinum was cultured aeroponically with different nutrient spraying intervals such as 5, 30, 60 and 240 min. The long spraying interval such as 240 min resulted in lower mass of root and shoot, shorter total root length with less tips and smaller surface area, compared to short interval of 5 min. Grown under the longest spraying interval of 240 min, M. crystallinumalso had significantly higher leaf dry matter content but lower leaf succulence. However, CAM acidity was undetectable for any plants. Although M. crystallinum grown under extended spraying intervals had higher photosynthetic pigments, they utilized lesser light energy and did not dissipate heat as effectively as those grown under 5 min. Compare to other shorter spraying intervals, photosynthetic gas exchange rates were significantly reduced under 240 min spraying interval, indicating signs of water deficit stress. Shoot nitrate, total reduced nitrogen, total soluble protein and Rubisco concentrations were similar for all plants. For phytochemicals and dietary minerals, plants grown under 240 min spraying interval had significantly higher values than the other plants. Therefore, drought does not result in the induction of CAM but regulates photosynthetic performance and enhances nutritional quality of M. crystallinum.


Assuntos
Secas , Fotossíntese/genética , Folhas de Planta/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/genética , Mesembryanthemum/química , Mesembryanthemum/metabolismo , Valor Nutritivo , Folhas de Planta/genética , Cloreto de Sódio/metabolismo
2.
Nat Struct Mol Biol ; 27(3): 281-287, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123388

RESUMO

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.


Assuntos
Proteínas de Bactérias/química , Halothiobacillus/química , Proteínas Intrinsicamente Desordenadas/química , Organelas/química , Ribulose-Bifosfato Carboxilase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ciclo do Carbono/fisiologia , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Halothiobacillus/genética , Halothiobacillus/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Organelas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática
3.
BMC Evol Biol ; 20(1): 11, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969115

RESUMO

BACKGROUND: The CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. RESULTS: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration. CONCLUSIONS: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.


Assuntos
Bromeliaceae/enzimologia , Carbono/metabolismo , Evolução Molecular , Orchidaceae/enzimologia , Ribulose-Bifosfato Carboxilase/genética , Adaptação Fisiológica , Isótopos de Carbono/metabolismo , Cinética , Fotossíntese , Filogenia , Folhas de Planta/genética , Subunidades Proteicas/metabolismo , Seleção Genética
4.
Proc Natl Acad Sci U S A ; 117(1): 381-387, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31848241

RESUMO

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acidithiobacillus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Chaperonas Moleculares/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Acidithiobacillus/genética , Acidithiobacillus/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Domínio Catalítico/genética , Cristalografia por Raios X , Ativação Enzimática , Ensaios Enzimáticos , Microscopia Eletrônica , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/ultraestrutura , Mutagênese Sítio-Dirigida , Multimerização Proteica , Estrutura Secundária de Proteína , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/ultraestrutura
5.
Proc Natl Acad Sci U S A ; 116(48): 24041-24048, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712424

RESUMO

During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO2-fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ∼2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO2-fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO2 assimilation rate, growth, and yield.


Assuntos
Oryza/enzimologia , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fotossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Domínios Proteicos , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética
6.
Genes (Basel) ; 10(11)2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671713

RESUMO

Allopolyploids are often faced with the challenge of maintaining well-coordination between nuclear and cytoplasmic genes inherited from different species. The synthetic allotetraploid Cucumis × hytivus is a useful model to explore cytonuclear coevolution. In this study, the sequences and expression of cytonuclear enzyme complex RuBisCO as well as its content and activity in C. × hytivus were compared to its parents to explore plastid-nuclear coevolution. The plastome-coded rbcL gene sequence was confirmed to be stable maternal inheritance, and parental copy of nuclear rbcS genes were both preserved in C. × hytivus. Thus, the maternal plastid may interact with the biparentally inherited rbcS alleles. The expression of the rbcS gene of C-homoeologs (paternal) was significantly higher than that of H-homoeologs (maternal) in C. × hytivus (HHCC). Protein interaction prediction analysis showed that the rbcL protein has stronger binding affinity to the paternal copy of rbcS protein than that of maternal copy in C. × hytivus, which might explain the transcriptional bias of the rbcS homoeologs. Moreover, both the activity and content of RuBisCO in C. × hytivus showed mid-parent heterosis. In summary, our results indicate a paternal transcriptional bias of the rbcS genes in C. × hytivus, and we found new nuclear-cytoplasmic combination may be one of the reasons for allopolyploids heterosis.


Assuntos
Cucumis/genética , Poliploidia , Ribulose-Bifosfato Carboxilase/genética , Alelos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quimera/genética , Citoplasma/metabolismo , Citosol/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Plastídeos/genética
7.
Proc Natl Acad Sci U S A ; 116(42): 20984-20990, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570616

RESUMO

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fotossíntese/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/química , Cianobactérias/genética , Cianobactérias/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Luz , NADP/química , NADP/metabolismo , Oxirredução/efeitos da radiação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo
8.
BMC Evol Biol ; 19(1): 192, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638910

RESUMO

BACKGROUND: The genus Ilex (Aquifoliaceae) has a near-cosmopolitan distribution in mesic habitats from tropical to temperate lowlands and in alpine forests. It has a high rate of hybridization and plastid capture, and comprises four geographically structured plastid groups. A previous study showed that the plastid rbcL gene, coding for the large subunit of Rubisco, has a particularly high rate of non-synonymous substitutions in Ilex, when compared with other plant lineages. This suggests a strong positive selection on rbcL, involved in yet unknown adaptations. We therefore investigated positive selection on rbcL in 240 Ilex sequences from across the global range. RESULTS: The rbcL gene shows a much higher rate of positive selection in Ilex than in any other plant lineage studied so far (> 3000 species) by tests in both PAML and SLR. Most positively selected residues are on the surface of the folded large subunit, suggesting interaction with other subunits and associated chaperones, and coevolution between positively selected residues is prevalent, indicating compensatory mutations to recover molecular stability. Coevolution between positively selected sites to restore global stability is common. CONCLUSIONS: This study has confirmed the predicted high incidence of positively selected residues in rbcL in Ilex, and shown that this is higher than in any other plant lineage studied so far. The causes and consequences of this high incidence are unclear, but it is probably associated with the similarly high incidence of hybridization and introgression in Ilex, even between distantly related lineages, resulting in large cytonuclear discordance in the phylogenies.


Assuntos
Ilex/genética , Ribulose-Bifosfato Carboxilase/genética , Seleção Genética , Sequência de Bases , Códon/genética , Evolução Molecular , Hibridização Genética , Modelos Moleculares , Filogenia , Ribulose-Bifosfato Carboxilase/química
9.
Int J Mol Sci ; 20(20)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658746

RESUMO

Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).


Assuntos
Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Ciclo do Carbono/genética , Ciclo do Carbono/fisiologia , Cianobactérias/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Cinética , Engenharia Metabólica , Modelos Moleculares , Mutação , Fotossíntese , Conformação Proteica , Ribulose-Bifosfato Carboxilase/química
10.
Biochem J ; 476(18): 2595-2606, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31471529

RESUMO

Rubisco activase (Rca) is a catalytic chaperone that remodels the active site, promotes the release of inhibitors and restores catalytic competence to Rubisco. Rca activity and its consequent effect on Rubisco activation and photosynthesis are modulated by changes to the chloroplast environment induced by fluctuations in light levels that reach the leaf, including redox status and adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio. The Triticum aestivum (wheat) genome encodes for three Rca protein isoforms: 1ß (42.7 kDa), 2ß (42.2 kDa) and 2α (46.0 kDa). The regulatory properties of these isoforms were characterised by measuring rates of Rubisco activation and ATP hydrolysis by purified recombinant Rca proteins in the presence of physiological ADP/ATP ratios. ATP hydrolysis by all three isoforms was sensitive to inhibition by increasing amounts of ADP in the assay. In contrast, Rubisco activation activity of Rca 2ß was insensitive to ADP inhibition, while Rca 1ß and 2α were inhibited. Two double and one quadruple site-directed mutants were designed to elucidate if differences in the amino acid sequences between Rca 1ß and 2ß could explain the differences in ADP sensitivity. Changing two amino acids in Rca 2ß to the corresponding residues in 1ß (T358K & Q362E) resulted in significant inhibition of Rubisco activation in presence of ADP. The results show that the wheat Rca isoforms differ in their regulatory properties and that amino acid changes in the C domain influence ADP sensitivity. Advances in the understanding of Rubisco regulation will aid efforts to improve the efficiency of photosynthetic CO2 assimilation.


Assuntos
Difosfato de Adenosina/química , Ribulose-Bifosfato Carboxilase/química , Triticum/enzimologia , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Ativação Enzimática/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação de Sentido Incorreto , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Triticum/genética
11.
Nat Plants ; 5(7): 715-721, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31285558

RESUMO

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.


Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Cloroplastos/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tabaco/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Celulase/análise , Celulase/genética , Cloroplastos/química , Cloroplastos/genética , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Tabaco/química , Tabaco/genética , Tabaco/metabolismo
12.
mBio ; 10(4)2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337726

RESUMO

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO2 into organic carbon in primary producers. All naturally occurring RubisCOs have low catalytic turnover rates and are inhibited by oxygen. Evolutionary adaptations of the enzyme and its host organisms to changing atmospheric oxygen concentrations provide an impetus to artificially evolve RubisCO variants under unnatural selective conditions. A RubisCO deletion strain of the nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus was previously used as a heterologous host for directed evolution and suppressor selection studies that led to the identification of a conserved hydrophobic region near the active site where amino acid substitutions selectively impacted the enzyme's sensitivity to O2 In this study, structural alignments, mutagenesis, suppressor selection, and growth complementation with R. capsulatus under anoxic or oxygenic conditions were used to analyze the importance of semiconserved residues in this region of Synechococcus RubisCO. RubisCO mutant substitutions were identified that provided superior CO2-dependent growth capabilities relative to the wild-type enzyme. Kinetic analyses of the mutant enzymes indicated that enhanced growth performance was traceable to differential interactions of the enzymes with CO2 and O2 Effective residue substitutions also appeared to be localized to two other conserved hydrophobic regions of the holoenzyme. Structural comparisons and similarities indicated that regions identified in this study may be targeted for improvement in RubisCOs from other sources, including crop plants.IMPORTANCE RubisCO catalysis has a significant impact on mitigating greenhouse gas accumulation and CO2 conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO2 fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO's kinetic properties to enhance growth in the presence of atmospheric O2 levels has been a longstanding goal. In this study, RubisCO variants with superior structure-functional properties were selected which resulted in enhanced growth of an autotrophic host organism (R. capsulatus), indicating that RubisCO function was indeed growth limiting. It is evident from these results that genetically engineered RubisCO with kinetically enhanced properties can positively impact growth rates in primary producers.


Assuntos
Rhodobacter capsulatus/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Synechococcus/genética , Dióxido de Carbono , Domínio Catalítico , Evolução Molecular Direcionada , Cinética , Mutação , Fotossíntese , Rhodobacter capsulatus/enzimologia , Synechococcus/enzimologia
13.
Plant Physiol Biochem ; 142: 163-172, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299598

RESUMO

C4 photosynthetic enzymes are present in C3 plants and participate in non-photosynthetic metabolism. Wheat spike bracts had a higher drought tolerance, photosynthesis and senesced later compared to the flag leaves under water deficit. This research was conducted to investigate the different response of primary carbon metabolism induced by C4 photosynthetic enzymes in wheat flag leaves and spike bracts including glumes and lemmas under water deficit. The activities of C4 photosynthetic enzymes and Ribulose bisphosphate carboxylase oxygenase (Rubisco), the expression of related genes and primary carbon metabolism contents were demonstrated in wheat flag leaves and spike bracts exposed to water deficit. Results showed that drought stress strongly inhibited wheat photosynthetic metabolism by decreasing Rubisco activity in flag leaves. The activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), phosphate dikinase (PPDK) and NADP- malic dehydrogenase (NADP-MDH) increased in wheat spike bracts under water deficit. Transcript levels of C4 photosynthetic genes in wheat spike bracts were higher under water deficit than that of control. Furthermore, the results indicated that drought stress induced changes in the contents of primary carbon metabolism including malate, oxaloacetic acid (OAA), citric, fumaric acid were organ-specific. In conclusion, the functions of C4 photosynthetic enzymes appear to be important for wheat spike bracts primary carbon metabolism and defence response under drought stress.


Assuntos
Carbono/metabolismo , Enzimas/genética , Enzimas/metabolismo , Triticum/fisiologia , Mapeamento Cromossômico , Cromossomos de Plantas , Secas , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/genética , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Triticum/metabolismo
14.
Int J Legal Med ; 133(5): 1351-1360, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31190289

RESUMO

The development of DNA barcoding method has given rise to a promising way of studying genetic taxonomy. Our previous study showed that pyrosequencing profile of 18S rDNA V7 hypervariable region can be used for identifying water sources without resolving the exact components of diatom colonies in water samples. In this continued study, we aimed to improve the established analysis method and to provide scientific evidence for forensic practices. A drowning animal model was set up by injecting mimic drowning fluid into the respiratory tract of the rabbit. In order to minimize the interference of animal DNA, the hypervariable region of chloroplast ribulose-1,5-bisphosphate carboxylase large unit gene (rbcL) was used as the pyrosequencing target region for the consistency analysis of plankton populations in tissues and water samples. After decoding the pyrosequencing profile of the targeted rbcL gene with the AdvISER-M-PYRO algorithm, the plankton colony that was inhaled into drowning animal lung tissue could be successfully traced back to the source of drowning fluid. Our data suggest that this method could be a reliable tool assisting forensic drowning site inference.


Assuntos
Código de Barras de DNA Taxonômico , Diatomáceas/classificação , Afogamento/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ribulose-Bifosfato Carboxilase/genética , Animais , Genética Forense , Patologia Legal , Modelos Animais , Reação em Cadeia da Polimerase , Coelhos , Sensibilidade e Especificidade
15.
ISME J ; 13(10): 2475-2488, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182769

RESUMO

Almost all the inorganic carbon on Earth is converted into biomass via the Calvin-Benson-Bassham (CBB) cycle. Here, the central carboxylation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which can be found in numerous primary producers including plants, algae, cyanobacteria, and many autotrophic bacteria. Although RubisCO possesses a crucial role in global biomass production, it is not a perfect catalyst. Therefore, research interest persists on accessing the full potential of yet unexplored RubisCOs. We recently developed an activity-based screen suited to seek active recombinant RubisCOs from the environment-independent of the native host's culturability. Here, we applied this screen to twenty pre-selected genomic fosmid clones from six cultured proteobacteria to demonstrate that a broad range of phylogenetically distinct RubisCOs can be targeted. We then screened 12,500 metagenomic fosmid clones from six distinct hydrothermal vents and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Fontes Hidrotermais/microbiologia , Proteobactérias/enzimologia , Ribulose-Bifosfato Carboxilase/metabolismo , Archaea/classificação , Archaea/genética , Archaea/isolamento & purificação , Proteínas Arqueais/genética , Processos Autotróficos , Proteínas de Bactérias/genética , Carbono/metabolismo , Metagenoma , Fotossíntese , Filogenia , Proteobactérias/classificação , Proteobactérias/genética , Proteobactérias/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/genética
16.
Mar Environ Res ; 149: 80-89, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181418

RESUMO

Photosynthesis in the seagrass Zostera muelleri remains poorly understood. We investigated the effect of reduced irradiance on the incorporation of 13C, gene expression of photosynthetic, photorespiratory and intermediates recycling genes as well as the enzymatic content and activity of Rubisco and PEPC within Z. muelleri. Following 48 h of reduced irradiance, we found that i) there was a ∼7 fold reduction in 13C incorporation in above ground tissue, ii) a significant down regulation of photosynthetic, photorespiratory and intermediates recycling genes and iii) no significant difference in enzyme activity and content. We propose that Z. muelleri is able to alter its physiology in order to reduce the amount of C lost through photorespiration to compensate for the reduced carbon assimilation as a result of reduced irradiance. In addition, the first estimated rate constant (Kcat) and maximum rates of carboxylation (Vcmax) of Rubisco is reported for the first time for Z. muelleri.


Assuntos
Dióxido de Carbono/metabolismo , Carbono/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Zosteraceae/metabolismo , Austrália , Isótopos de Carbono/análise , Expressão Gênica , Luz , Fosfoenolpiruvato Carboxilase/genética , Fotossíntese/genética , Fotossíntese/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Zosteraceae/genética
17.
J Plant Physiol ; 237: 12-20, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999073

RESUMO

Functions of domains or motifs, which are encoded by the transit peptide (TP) of the precursor of the small subunit of Rubisco (prSSU), have been investigated intensively in dicots. Functional characterization of the prSSU TP, however, is still understudied in maize. In this study, we found that the TP of maize prSSU1 did not function fully in chloroplast targeting in Arabidopsis or vice versa, indicating the divergent function of TPs in chloroplast targeting between maize and Arabidopsis. Through deletion or substitution assays, we found that the N-terminal region of maize or Arabidopsis prSSU1 was necessary and sufficient for importing specifically the fused-green fluorescent protein (GFP) into each corresponding chloroplast. Finally, we found that the first-five amino acids and MM motif in the N-terminal domain of the maize TP played an essential role in maize chloroplast targeting. Thus, our analyses demonstrate that the N-terminal domain of the prSSU1 TP is the key determinant in chloroplast targeting between maize and Arabidopsis. Our study highlights the unique properties of the maize prSSU1 TP in chloroplast targeting, thus helping to understand the role of N-terminal domain in chloroplast targeting across species. It will help to manipulate chloroplast transit peptides (cTPs) for crop bioengineering.


Assuntos
Arabidopsis/genética , Proteínas de Cloroplastos/genética , Sinais Direcionadores de Proteínas/genética , Ribulose-Bifosfato Carboxilase/genética , Zea mays/genética , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Zea mays/enzimologia , Zea mays/metabolismo
18.
J Biosci Bioeng ; 128(3): 302-306, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30987875

RESUMO

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1PsSTQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (ΔglcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1PsSTQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms.


Assuntos
Escherichia coli , Glicolatos/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Ribulose-Bifosfato Carboxilase/fisiologia , Ribulosefosfatos/metabolismo , Dióxido de Carbono/metabolismo , Clonagem Molecular/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Engenharia Metabólica/métodos , Organismos Geneticamente Modificados , Fotossíntese/fisiologia , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo
19.
Metab Eng ; 54: 222-231, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31029860

RESUMO

Cyanobacterial carboxysomes encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Genetic deletion of the major structural proteins encoded within the ccm operon in Synechococcus sp. PCC 7002 (ΔccmKLMN) disrupts carboxysome formation and significantly affects cellular physiology. Here we employed both metabolite pool size analysis and isotopically nonstationary metabolic flux analysis (INST-MFA) to examine metabolic regulation in cells lacking carboxysomes. Under high CO2 environments (1%), the ΔccmKLMN mutant could recover growth and had a similar central flux distribution as the control strain, with the exceptions of moderately decreased photosynthesis and elevated biomass protein content and photorespiration activity. Metabolite analyses indicated that the ΔccmKLMN strain had significantly larger pool sizes of pyruvate (>18 folds), UDPG (uridine diphosphate glucose), and aspartate as well as higher levels of secreted organic acids (e.g., malate and succinate). These results suggest that the ΔccmKLMN mutant is able to largely maintain a fluxome similar to the control strain by changing in intracellular metabolite concentrations and metabolite overflows under optimal growth conditions. When CO2 was insufficient (0.2%), provision of acetate moderately promoted mutant growth. Interestingly, the removal of microcompartments may loosen the flux network and promote RuBisCO side-reactions, facilitating redirection of central metabolites to competing pathways (i.e., pyruvate to heterologous lactate production). This study provides important insights into metabolic regulation via enzyme compartmentation and cyanobacterial compensatory responses.


Assuntos
Proteínas de Bactérias , Análise do Fluxo Metabólico , Mutação , Óperon , Fotossíntese/genética , Ribulose-Bifosfato Carboxilase , Synechococcus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Synechococcus/enzimologia , Synechococcus/genética
20.
ACS Synth Biol ; 8(5): 1219-1223, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30973704

RESUMO

The cyanobacterium Synechococcus elongatus PCC 7942 is a potential photosynthetic cell-factory. In this study, two native promoters from S. elongatus PCC 7942 driving the expression of abundant cyanobacterial proteins phycocyanin (P cpcB7942) and RuBisCO (P rbc7942) were characterized in relation to their sequence features, expression levels, diurnal behavior, and regulation by light and CO2, major abiotic factors important for cyanobacterial growth. P cpcB7942 was repressed under high light intensity, but cultivation at higher CO2 concentration was able to recover promoter activity. On the other hand, P rbc7942 was repressed by elevated CO2 with a negative regulatory region between 300 and 225 bp. Removal of this region flipped the effect of CO2 with Rbc225 being activated only at high CO2 concentration, besides leading to the loss of circadian rhythm. The results from this study on promoter features and regulation will help expand the repertoire of tools for pathway engineering in cyanobacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Synechococcus/genética , Proteínas de Bactérias/genética , Dióxido de Carbono/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Genes Reporter , Luz , Ficocianina/genética , Regiões Promotoras Genéticas , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/crescimento & desenvolvimento
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