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1.
Nat Commun ; 12(1): 5150, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446715

RESUMO

Recent studies have revealed the prevalence and biological significance of guanidine metabolism in nature. However, the metabolic pathways used by microbes to degrade guanidine or mitigate its toxicity have not been widely studied. Here, via comparative proteomics and subsequent experimental validation, we demonstrate that Sll1077, previously annotated as an agmatinase enzyme in the model cyanobacterium Synechocystis sp. PCC 6803, is more likely a guanidinase as it can break down guanidine rather than agmatine into urea and ammonium. The model cyanobacterium Synechococcus elongatus PCC 7942 strain engineered to express the bacterial ethylene-forming enzyme (EFE) exhibits unstable ethylene production due to toxicity and genomic instability induced by accumulation of the EFE-byproduct guanidine. Co-expression of EFE and Sll1077 significantly enhances genomic stability and enables the resulting strain to achieve sustained high-level ethylene production. These findings expand our knowledge of natural guanidine degradation pathways and demonstrate their biotechnological application to support ethylene bioproduction.


Assuntos
Proteínas de Bactérias/metabolismo , Etilenos/biossíntese , Instabilidade Genômica , Guanidina/metabolismo , Synechococcus/genética , Synechococcus/metabolismo , Synechocystis/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Synechocystis/genética
2.
Sci Rep ; 11(1): 17131, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429477

RESUMO

A unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses a unique tricarboxylic acid (TCA) cycle, wherein the intracellular citrate levels are approximately 1.5-10 times higher than the levels of other TCA cycle metabolite. Aconitase catalyses the reversible isomerisation of citrate and isocitrate. Herein, we biochemically analysed Synechocystis sp. PCC 6803 aconitase (SyAcnB), using citrate and isocitrate as the substrates. We observed that the activity of SyAcnB for citrate was highest at pH 7.7 and 45 °C and for isocitrate at pH 8.0 and 53 °C. The Km value of SyAcnB for citrate was higher than that for isocitrate under the same conditions. The Km value of SyAcnB for isocitrate was 3.6-fold higher than the reported Km values of isocitrate dehydrogenase for isocitrate. Therefore, we suggest that citrate accumulation depends on the enzyme kinetics of SyAcnB, and 2-oxoglutarate production depends on the chemical equilibrium in this cyanobacterium.


Assuntos
Aconitato Hidratase/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Cítrico/metabolismo , Synechocystis/enzimologia , Ácido Cítrico/análogos & derivados , Concentração de Íons de Hidrogênio , Isomerismo , Cinética , Especificidade por Substrato , Synechocystis/metabolismo , Temperatura
3.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
4.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34039816

RESUMO

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains-Synechocystis sp. PCC6803 and Synechococcus sp. WH7803-were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.


Assuntos
Proteínas de Bactérias/metabolismo , Sulfato Adenililtransferase/metabolismo , Synechococcus/enzimologia , Synechococcus/crescimento & desenvolvimento , Synechocystis/enzimologia , Synechocystis/crescimento & desenvolvimento , Compostos de Amônio/metabolismo , Proteínas de Bactérias/genética , Sulfato Adenililtransferase/genética , Sulfatos/metabolismo , Synechococcus/genética , Synechocystis/genética
5.
Plant Sci ; 304: 110798, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568297

RESUMO

Cyanobacterial type I NADH dehydrogenase (NDH-1) is involved in various bioenergetic reactions including respiration, cyclic electron transport (CET), and CO2 uptake. The role of NDH-1 is usually minor under normal growth conditions and becomes important under stress conditions. However, in our previous study, flux balance analysis (FBA) simulation predicted that the drive of NDH-1 as CET pathway with a photosystem (PS) I/PSII excitation ratio around 1.0 contributes to achieving an optimal specific growth rate. In this study, to experimentally elucidate the predicted functions of NDH-1, first, we measured the PSI/PSII excitation ratios of Synechocystis sp. PCC 6803 grown under four types of spectral light conditions. The specific growth rate was the highest and PSI/PSII excitation ratio was with 0.88 under the single-peak light at 630 nm (Red1). Considering this measured excitation ratios, FBA simulation predicted that NDH-1-dependent electron transport was the major pathway under Red1. Moreover, in actual culture, an NDH-1 deletion strain had slower growth rate than that of wild type only under Red1 light condition. Therefore, we experimentally demonstrated that NDH-1 plays an important role under optimal light conditions such as Red1 light, where Synechocystis exhibits the highest specific growth rate and PSI/PSII excitation ratio of around 1.0.


Assuntos
Proteínas de Bactérias/fisiologia , Complexo I de Transporte de Elétrons/fisiologia , Ficobilissomas/farmacologia , Synechocystis/enzimologia , Proteínas de Bactérias/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Luz , Consumo de Oxigênio , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/crescimento & desenvolvimento , Synechocystis/efeitos da radiação
6.
Biochem Biophys Res Commun ; 540: 16-21, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33429195

RESUMO

Polyphosphate, which is ubiquitous in cells in nature, is involved in a myriad of cellular functions, and has been recently focused on its metabolism related with microbial acclimation to phosphorus-source fluctuation. In view of the ecological importance of cyanobacteria as the primary producers, this study investigated the responsibility of polyphosphate metabolism for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803, with the use of a disruptant (Δppx) as to the gene of exopolyphosphatase that is responsible for polyphosphate degradation. Δppx was similar to the wild type in the cellular content of polyphosphate to show no defect in cell growth under phosphorus-replete conditions. However, under phosphorus-starved conditions, Δppx cells were defective in a phosphorus-starvation dependent decrease of polyphosphate to show deleterious phenotypes as to their survival and the stabilization of the photosystem complexes. These results demonstrated some crucial role of exopolyphosphatase to degrade polyP in the acclimation of cyanobacterial cells to phosphorus-starved conditions. Besides, it was found that ppx expression is induced in Synechocystis cells in response to phosphorus starvation through the action of the two-component system, SphS and SphR, in the phosphate regulon. The information will be a foundation for a fuller understanding of the process of cyanobacterial acclimation to phosphorus fluctuation.


Assuntos
Hidrolases Anidrido Ácido/genética , Fósforo/deficiência , Fósforo/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Aclimatação , Proteínas de Bactérias/genética , Viabilidade Microbiana , Polifosfatos/metabolismo , Regulon , Synechocystis/citologia , Synechocystis/enzimologia
7.
Appl Biochem Biotechnol ; 193(3): 687-716, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33159456

RESUMO

Cyanobacterium Synechocystis sp. PCC 6803, a popular model organism for researches in photosynthesis and biofuel production, contains plant-like photosynthetic machineries which significantly contribute to global carbon fixation. There are 12 eukaryotic-type Ser/Thr kinases (SpkA-L) and 49 His kinases (Hik1-49) of two-component systems in the genome of Synechocystis sp. PCC 6803. They are the key regulators in sensing and transmitting stimuli including light- and glucose-mediate signal transduction. Proteomic studies were able to identify all the kinases. The majority of kinases no matter whether they have a predicted transmembrane domain were identified in the membrane fractions. Six Ser/Thr kinases (SpkA-D, F and G) and ten His kinases (Hik4, 12, 14, 21, 26-27, 29, 36, 43, and 46) were identified to have one or more of the three types of post-translational modifications: phosphorylation, acetylation, and thiol oxidation. Interestingly, SpkG has the phosphorylatable threonine residue that was aligned with the phosphorylated threonine residue in the activation loop of human CDK7, demonstrating conserved phosphorylation between cyanobacterial and human kinases. Transcriptomics and proteomics revealed differential expression of the kinases in heterotrophic and photoheterotrophic compared with photoautotrophic conditions, indicating their roles in regulating the growth modes of cyanobacteria. In summary, this review focuses on the discussions on post-transcriptional modifications, transcriptomic, and proteomic studies of Ser/Thr and His kinases. This together with our published review in 2019 present a complete story of an overview of sequences, domain architectures, and biochemical and physiological functions of cyanobacterial kinases with adequate details in the context of high throughput systems. We also emphasize the importance of discovering upstream molecules and substrates to understand the exact functions of the kinases in vivo. As an attempt, a model is proposed in which Hik31, His33, Sll1334, and IcfG are hypothesized to be critical for switching between autotrophic and heterotrophic modes based on the results from the phenotypes of the gene knockout strains combined with their post-translational modifications, and gene expression profiles.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Synechocystis/enzimologia , Fosforilação
8.
Angew Chem Int Ed Engl ; 60(7): 3679-3684, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33141478

RESUMO

A mirror-image strategy, that is, symmetry analysis of the substrate-binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis sp. PCC6803 towards 3-isobutyl glutaronitrile (1 a). Exchange of these two residues resulted in the enantiopreference inversion (S, 90 % ee to R, 47 % ee). By further reshaping the substrate-binding pocket via routine site-saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99 % ee, R) was obtained. The mutant enzyme was applied in the preparation of optically pure (R)-3-isobutyl-4-cyanobutanoic acid ((R)-2 a) and showed similar stereopreference inversion towards a series of 3-substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups.


Assuntos
Aminoidrolases/metabolismo , Nitrilas/metabolismo , Aminoidrolases/genética , Sítios de Ligação , Biocatálise , Hidrólise , Estrutura Molecular , Nitrilas/química , Estereoisomerismo , Synechocystis/enzimologia
9.
Environ Microbiol ; 23(2): 559-571, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908125

RESUMO

Protein quality control pathways require AAA+ proteases, such as Clp and Lon. Lon protease maintains UmuD, an important component of the error-prone DNA repair polymerase (Pol V), at very low levels in E. coli. Most members of the phylum Cyanobacteria lack Lon (including the model cyanobacterium, Synechocystis sp. PCC6803), so maintenance of UmuD at low levels must employ different proteases. We demonstrate that the first 19 residues from the N-terminus of UmuD (Sug1-19 ) fused to a reporter protein are adequate to trigger complete proteolysis and that mutation of a single leucine residue (L6) to aspartic acid inhibits proteolysis. This process appears to follow the N-end rule and is mediated by ClpA/P protease and the ClpS adaptor. Additionally, mutations of arginine residues in the Sug1-19 tag suggest that the ClpX/P pathway also plays a role in proteolysis. We propose that there is a dual degron at the N-terminus of the UmuD protein in Synechocystis sp. PCC6803, which is distinct from the degron required for degradation of UmuD in E. coli. The use of two proteolysis pathways to tune levels of UmuD might reflect how a photosynthetic organism responds to multiple environmental stressors.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Synechocystis/enzimologia , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Mutação , Proteólise , Synechocystis/química , Synechocystis/genética , Synechocystis/metabolismo
10.
Biochemistry ; 59(51): 4864-4872, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33319991

RESUMO

The S3 state is the last semi-stable state in the water splitting reaction that is catalyzed by the Mn4O5Ca cluster that makes up the oxygen-evolving complex (OEC) of photosystem II (PSII). Recent high-field/frequency (95 GHz) electron paramagnetic resonance (EPR) studies of PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus have found broadened signals induced by chemical modification of the S3 state. These signals are ascribed to an S3 form that contains a five-coordinate MnIV center bridged to a cuboidal MnIV3O4Ca unit. High-resolution X-ray free-electron laser studies of the S3 state have observed the OEC with all-octahedrally coordinated MnIV in what is described as an open cuboid-like cluster. No five-coordinate MnIV centers have been reported in these S3 state structures. Here, we report high-field/frequency (130 GHz) pulse EPR of the S3 state in Synechocystis sp. PCC 6803 PSII as isolated in the presence of glycerol. The S3 state of PSII from Synechocystis exhibits multiple broadened forms (≈69% of the total signal) similar to those seen in the chemically modified S3 centers from T. elongatus. Field-dependent ELDOR-detected nuclear magnetic resonance resolves two classes of 55Mn nuclear spin transitions: one class with small hyperfine couplings (|A| ≈ 1-7 MHz) and another with larger hyperfine couplings (|A| ≈ 100 MHz). These results are consistent with an all-MnIV4 open cubane structure of the S3 state and suggest that the broadened S3 signals arise from a perturbation of Mn4A and/or Mn3B, possibly induced by the presence of glycerol in the as-isolated Synechocystis PSII.


Assuntos
Complexo de Proteína do Fotossistema II/química , Synechocystis/enzimologia , Crioprotetores/química , Espectroscopia de Ressonância de Spin Eletrônica , Glicerol/química , Manganês/química , Oxirredução , Oxigênio/química
11.
Sci Rep ; 10(1): 22018, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328526

RESUMO

Phosphoketolase (PKET) pathway is predominant in cyanobacteria (around 98%) but current opinion is that it is virtually inactive under autotrophic ambient CO2 condition (AC-auto). This creates an evolutionary paradox due to the existence of PKET pathway in obligatory photoautotrophs. We aim to answer the paradox with the aid of bioinformatic analysis along with metabolic, transcriptomic, fluxomic and mutant data integrated into a multi-level kinetic model. We discussed the problems linked to neglected isozyme, pket2 (sll0529) and inconsistencies towards the explanation of residual flux via PKET pathway in the case of silenced pket1 (slr0453) in Synechocystis sp. PCC 6803. Our in silico analysis showed: (1) 17% flux reduction via RuBisCO for Δpket1 under AC-auto, (2) 11.2-14.3% growth decrease for Δpket2 in turbulent AC-auto, and (3) flux via PKET pathway reaching up to 252% of the flux via phosphoglycerate mutase under AC-auto. All results imply that PKET pathway plays a crucial role under AC-auto by mitigating the decarboxylation occurring in OPP pathway and conversion of pyruvate to acetyl CoA linked to EMP glycolysis under the carbon scarce environment. Finally, our model predicted that PKETs have low affinity to S7P as a substrate.


Assuntos
Aldeído Liases/metabolismo , Redes e Vias Metabólicas , Synechocystis/enzimologia , Carbono/metabolismo , Simulação por Computador , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Análise do Fluxo Metabólico , Filogenia , Especificidade por Substrato , Fosfatos Açúcares/metabolismo , Synechocystis/genética
12.
Genomics Proteomics Bioinformatics ; 18(3): 289-304, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-33130100

RESUMO

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.


Assuntos
Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Metiltransferases/metabolismo , Fotossíntese , Processamento de Proteína Pós-Traducional , Synechocystis/enzimologia , Proteínas de Bactérias/química , Lisina/química , Metiltransferases/química , Synechocystis/crescimento & desenvolvimento
13.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 11): 524-535, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33135671

RESUMO

The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Šresolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, ß = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.


Assuntos
Francisella tularensis/enzimologia , Frutose-Bifosfatase/química , Frutose-Bifosfatase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/isolamento & purificação , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Estrutura Quaternária de Proteína , Synechocystis/enzimologia
14.
Biochimie ; 179: 46-53, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32946991

RESUMO

Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C-C bonds in the fatty acids (FAs) producing unsaturated CC double bonds that markedly change the properties of biological membranes. FADs are highly specific towards their acyl substrates, the position and configuration of the introduced double bonds. The double bond positioning of soluble acyl-carrier-protein Δ9-FADs was determined relative to the carboxyl end of a FA. Similar mode was suggested for the acyl-lipid Δ12-FADs (also known as ω6-FADs), however, their exact counting order remain unknown. Here we used monounsaturated odd- (17:1Δ10) and even-chain (18:1Δ11) FAs to show that acyl-lipid Δ12-FADs of, at least, two cyanobacterial species, Gloeobacter violaceus and Synechocystis sp. strain PCC 6803, use neither end of the fatty acid (Δ or ω) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of a FA chain length.


Assuntos
Carbono/química , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/provisão & distribuição , Ácidos Graxos Monoinsaturados/química , Carbono/metabolismo , Cianobactérias/química , Cianobactérias/enzimologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Monoinsaturados/isolamento & purificação , Ácidos Graxos Monoinsaturados/metabolismo , Galactolipídeos/análise , Glicolipídeos/análise , Metabolismo dos Lipídeos , Fosfatidilgliceróis/análise , Espectrometria de Massas por Ionização por Electrospray , Synechococcus/química , Synechococcus/enzimologia , Synechocystis/química , Synechocystis/enzimologia
15.
DNA Repair (Amst) ; 95: 102942, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32795961

RESUMO

Cyanobacterial species, Anabaena/Nostoc and Chroococcidiopsis are highly radio-resistant indicating the presence of a robust DNA repair system. However, unlike the establishment of multiple DNA repair pathways in the radio-resistant Deinococcus, research on DNA repair in cyanobacteria has lagged far behind. Being ancient organisms, it is likely that the DNA repair mechanisms have evolved from cyanobacteria to the modern day bacteria. This review focuses on identifying and collating information on the major DNA repair proteins in cyanobacteria including re-annotation of recR and ndk, using Anabaena/Nostoc sp. strain PCC7120 as a model organism. Unlike most other bacteria, the DNA repair genes of cyanobacteria are not clustered in operons. Though the functional characterisation of most DNA repair proteins is lacking in cyanobacteria, a bioinformatic approach using sequences of DNA repair proteins from Anabaena PCC7120, has helped identify the possible protein-protein interactions, and build probable pathways of double strand break (DSB) repair. The emerging picture can be used as a guide to discern the biochemical and physiological roles of the different DNA repair proteins in Anabaena or Synechocystis, which can be manipulated genetically and establish the different DNA repair pathways in cyanobacteria, and their evolution with time.


Assuntos
Cianobactérias/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Anabaena/enzimologia , Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Cianobactérias/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Bacteriano/metabolismo , Synechocystis/enzimologia , Synechocystis/genética , Synechocystis/metabolismo
16.
ACS Chem Biol ; 15(8): 2281-2288, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32786290

RESUMO

Microbes are essential to the global ecosystem, but undesirable microbial growth causes issues ranging from food spoilage and infectious diseases to harmful cyanobacterial blooms. The use of chemicals to control microbial growth has achieved significant success, while specific roles for a majority of essential genes in growth control remain unexplored. Here, we show the growth inhibition of cyanobacterial species by targeting an essential enzyme for the biosynthesis of branched-chain amino acids. Specifically, we report the biochemical, genetic, and structural characterization of dihydroxyacid dehydratase from the model cyanobacterium Synechocystis sp. PCC 6803 (SnDHAD). Our studies suggest that SnDHAD is an oxygen-stable enzyme containing a [2Fe-2S] cluster. Furthermore, we demonstrate that SnDHAD is selectively inhibited in vitro and in vivo by the natural product aspterric acid, which also inhibits the growth of representative bloom-forming Microcystis and Anabaena strains but has minimal effects on microbial pathogens with [4Fe-4S] containing DHADs. This study suggests DHADs as a promising target for the precise growth control of microbes and highlights the exploration of other untargeted essential genes for microbial management.


Assuntos
Hidroliases/metabolismo , Synechocystis/enzimologia , Synechocystis/crescimento & desenvolvimento , Domínio Catalítico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Ferro-Enxofre/metabolismo , Mutação , Oxigênio/metabolismo
17.
Appl Biochem Biotechnol ; 192(4): 1346-1367, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32767175

RESUMO

Alcohol dehydrogenase is one of the most critical enzymes in the production of ethanol and butanol. Synechocystis sp. PCC 6803 is a model cyanobacterium organism that is able to produce alcohols through its autotrophic energy production system. In spite of the high potential for biofuel production by this bacteria, the structure of its alcohol dehydrogenase has not been subjected to in-depth studies. The current study was aimed to analyze the molecular model for alcohol dehydrogenase of Synechocystis sp. PCC 6803 and scrutinize the interactions of different chemicals, including substrates and coenzymes. Also, the phylogenetic tree was provided to investigate the relation between different sources. The results indicated that alcohol dehydrogenase of Synechocystis sp. PCC 6803 has a different sequence compared with other Alcohol dehydrogenases (ADHs) of cyanobacterial family members. Verification of the homology model using Ramachandran plot by PROCHECK indicated that all of the residues are in favored or allowed regions of the plot. This enzyme has two Zn ions in its structure which is very similar to the other Zn-dependent ADHs. Docking studies suggest that this enzyme could have more active sites for different substrates. In addition, this enzyme has more affinity to NADH as a cofactor and sinapaldehyde as a substrate compared with the other cofactor and substrates.


Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Biocombustíveis , Simulação por Computador , Synechocystis/enzimologia , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica
18.
Proc Natl Acad Sci U S A ; 117(33): 19914-19925, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747548

RESUMO

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward ß-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce ß-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/química , Carotenoides/metabolismo , Dioxigenases/química , Archaea/química , Archaea/classificação , Archaea/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carotenoides/química , Catálise , Domínio Catalítico , Dioxigenases/genética , Dioxigenases/metabolismo , Especificidade por Substrato , Synechocystis/química , Synechocystis/enzimologia , Synechocystis/genética
19.
mBio ; 11(4)2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753490

RESUMO

Methylglyoxal (MG) is a detrimental metabolic by-product that threatens most organisms (in humans MG causes diabetes). MG is predominantly detoxified by the glyoxalase pathway. This process begins with the conjugation of MG with glutathione (GSH), yielding a hemithioacetal product that is subsequently transformed by the glyoxalase enzymes into d-lactate and GSH. MG has been overlooked in photosynthetic organisms, although they inevitably produce it not only by the catabolism of sugars, lipids, and amino acids, as do heterotrophic organisms, but also by their active photoautotrophic metabolism. This is especially true for cyanobacteria that are regarded as having developed photosynthesis and GSH-dependent enzymes to detoxify the reactive oxygen species produced by their photosynthesis (CO2 assimilation) and respiration (glucose catabolism), which they perform in the same cell compartment. In this study, we used a combination of in vivo and in vitro approaches to characterize a logical, but as yet never described, link between MG detoxification and a (prokaryotic) representative of the evolutionarily conserved glutathione transferase (GST) detoxification enzymes. We show that the Sll0067 GST of the model cyanobacterium Synechocystis sp. strain PCC 6803 plays a prominent role in MG tolerance and detoxification, unlike the other five GSTs of this organism. Sll0067 catalyzes the conjugation of MG with GSH to initiate its elimination driven by glyoxalases. These results are novel because the conjugation of MG with GSH is always described as nonenzymatic. They will certainly stimulate the analysis of Sll0067 orthologs from other organisms with possible impacts on human health (development of biomarkers or drugs) and/or agriculture.IMPORTANCE In most organisms, methylglyoxal (MG), a toxic metabolite by-product that causes diabetes in humans, is predominantly detoxified by the glyoxalase enzymes. This process begins with the so-called "spontaneous" conjugation of MG with the cytoprotectant metabolite glutathione (GSH). In this study, we unravel a logical, but as yet unsuspected, link between MG detoxification and a (prokaryotic) representative of the ubiquitous glutathione transferase (GST) enzymes. We show that a GST of a model cyanobacterium plays a prominent role in the detoxification of MG in catalyzing its conjugation with GSH. This finding is important because this reaction, always regarded as nonenzymatic, could exist in plants and/or human and thus have an impact on agriculture and/or human health.


Assuntos
Glutationa Transferase/metabolismo , Aldeído Pirúvico/metabolismo , Synechocystis/enzimologia , Biocatálise , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo
20.
Int J Biol Macromol ; 162: 1054-1063, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32603730

RESUMO

One popular and relevant proposed function for cyanobacterial hemoglobin (Synechocystis Hb) is anaerobic nitrite reductase in vivo. During such reduction reactions, the hexacoordinated heme iron atom of SynHb is oxidized from the ferrous (Fe+2) to ferric (Fe+3) state and prevent damage by limiting the concentration of toxic metabolites such as nitrite. In order to perform these functions in vivo, there must be a mechanism that converts inactive Fe+3-SynHb back to the active Fe+2-SynHb to accomplish the nitrite reductase function. Here, we report a cognate reductase protein for Synechocystis hemoglobin which can reduce the Fe+3-SynHb to Fe+2-SynHb, thus lending a support to the proposed nitrite reductase function. This reductase is also able to reduce pentacoordinate Hbs such as myoglobin but with lower affinity compared to hexacoordinate SynHb. Insilico model of reductase protein-cyanobacterial hemoglobin complex revealed that the heme active site of Hb faces the catalytic center of the reductase protein and several amino acids in the interface interacts non-covalently thus favoring their interaction. Overall, our in vitro study provides the basic foundation for the understanding of the specific molecular mechanism of action and interaction of the SynHb reductase protein, which need to be investigated in further detail.


Assuntos
Proteínas de Bactérias/química , Hemoglobinas/química , Modelos Moleculares , Oxirredutases/química , Synechocystis/enzimologia , Proteínas de Bactérias/genética , Oxirredutases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Synechocystis/genética
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