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1.
FEMS Microbiol Ecol ; 97(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33512460

RESUMO

Meromictic lakes are interesting ecosystems to study anaerobic microorganisms due their permanent stratification allowing the formation of a stable anoxic environment. The crenogenic meromictic Lake Cadagno harbors an important community of anoxygenic phototrophic sulfur bacteria responsible for almost half of its total productivity. Besides their ability to fix CO2 through photosynthesis, these microorganisms also showed high rates of dark carbon fixation via chemosyntesis. Here, we grew in pure cultures three populations of anoxygenic phototrophic sulfur bacteria previously isolated from the lake, accounting for 72.8% of the total microbial community and exibiting different phenotypes: (1) the motile, large-celled purple sulfur bacterium (PSB) Chromatium okenii, (2) the small-celled PSB Thiodictyon syntrophicum and (3) the green sulfur bacterium (GSB) Chlorobium phaeobacteroides. We measured their ability to fix CO2 through photo- and chemo-synthesis, both in situ in the lake and in laboratory under different incubation conditions. We also evaluated the efficiency and velocity of H2S photo-oxidation, an important reaction in the anoxygenic photosynthesis process. Our results confirm that phototrophic sulfur bacteria strongly fix CO2 in the presence of light and that oxygen increases chemosynthesis at night, in laboratory conditions. Moreover, substancial differences were displayed between the three selected populations in terms of activity and abundance.


Assuntos
Ecossistema , Lagos , Chlorobium , Chromatium , Enxofre
2.
Bioorg Chem ; 102: 104111, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32738567

RESUMO

Chlorosomes in the green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, e, or f molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, in vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives bearing a methyl-, ethyl- or propyl-esterifying group and its methyl ester analogs with additional alkyl and hydroxy groups at the C132-position were examined using the BciC enzyme. The BciC-catalyzed reaction activity for the C132-methoxycarbonylated substrate was comparable to that for the ethoxycarbonylated compound; however, depropoxycarbonylation did not proceed. The BciC enzymatic demethoxycarbonylation of zinc methyl C132-alkylated pheophorbides a was gradually suppressed with the elongation of the alkyl chain and finally became inactive for the propyl substrate. The reaction of the C132-hydroxylated substrate (allomer) was accelerated compared to that of the C132-methyl analog possessing a similar steric size, and gave the corresponding C132-oxo product via further air-oxidation. All of the abovementioned enzymatic reactions occurred for one of the C132-epimers with the same configuration as in chlorophyllide a. The above substrate specificities and product distributions indicated the stereochemistry and size of the BciC enzymatic active site (pocket).


Assuntos
Proteínas de Bactérias/metabolismo , Chlorobium/enzimologia , Clorofila A/metabolismo , Complexos de Coordenação/metabolismo , Zinco/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clorofila A/química , Complexos de Coordenação/química , Relação Dose-Resposta a Droga , Conformação Molecular , Relação Estrutura-Atividade , Especificidade por Substrato , Zinco/química
3.
Protein Sci ; 28(10): 1840-1849, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31411782

RESUMO

ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY, full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.


Assuntos
ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Sítios de Ligação , Biocatálise , Chlorobium/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida
4.
Environ Microbiol ; 21(10): 3896-3908, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299137

RESUMO

Sulphide-driven anoxygenic photosynthesis is an ancient microbial metabolism that contributes significantly to inorganic carbon fixation in stratified, sulphidic water bodies. Methods commonly applied to quantify inorganic carbon fixation by anoxygenic phototrophs, however, cannot resolve the contributions of distinct microbial populations to the overall process. We implemented a straightforward workflow, consisting of radioisotope labelling and flow cytometric cell sorting based on the distinct autofluorescence of bacterial photopigments, to discriminate and quantify contributions of co-occurring anoxygenic phototrophic populations to in situ inorganic carbon fixation in environmental samples. This allowed us to assign 89.3% ± 7.6% of daytime inorganic carbon fixation by anoxygenic phototrophs in Lake Rogoznica (Croatia) to an abundant chemocline-dwelling population of green sulphur bacteria (dominated by Chlorobium phaeobacteroides), whereas the co-occurring purple sulphur bacteria (Halochromatium sp.) contributed only 1.8% ± 1.4%. Furthermore, we obtained two metagenome assembled genomes of green sulphur bacteria and one of a purple sulphur bacterium which provides the first genomic insights into the genus Halochromatium, confirming its high metabolic flexibility and physiological potential for mixo- and heterotrophic growth.


Assuntos
Chlorobium/metabolismo , Chromatiaceae/metabolismo , Lagos/microbiologia , Sulfetos/metabolismo , Enxofre/metabolismo , Ciclo do Carbono , Chlorobium/isolamento & purificação , Chromatiaceae/isolamento & purificação , Croácia , Fotossíntese , Água do Mar/microbiologia
5.
Curr Microbiol ; 76(11): 1290-1297, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31321468

RESUMO

The Lipid A component of the outer membrane of Gram-negative bacteria is an integral part of the permeability barrier known as LPS, which actively prevents the uptake of bactericidal compounds. It is clinically very significant, as it is known to elicit a strong immune response in the humans, through the TLR4 complex. The Lipid A species are synthesized through a highly conserved multistep biosynthetic pathway. The final step is catalyzed by acyltransferases of the HtrB/MsbB family, which are members of a superfamily of enzymes, present in all domains of life with important roles to play in various biological processes. The investigation of a putative dual functioning enzyme which can add both laurate and myristate residues to the (Kdo)2-lipid IVA (precursor of Lipid A) would give a snapshot into the versatility of substrates that these enzymes catalyze. In this study we have cloned and purified to homogeneity, such a putative dual functional acyltransferase from Chlorobium tepidum, and attempted to study the enzyme in more details in terms of its sequence and structural aspects, as it lacks conserved residues with other enzymes of the same family.


Assuntos
Aciltransferases/química , Proteínas de Bactérias/química , Membrana Celular/enzimologia , Chlorobium/enzimologia , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorobium/química , Chlorobium/genética , Chlorobium/metabolismo , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeo A/análogos & derivados , Lipídeo A/metabolismo , Filogenia , Alinhamento de Sequência
6.
J Am Chem Soc ; 141(17): 6906-6914, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30943021

RESUMO

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-α-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria.


Assuntos
Biocatálise , Ergotioneína/biossíntese , Sulfurtransferases/química , Domínio Catalítico/genética , Chlorobium/enzimologia , Cristalografia por Raios X , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Sulfurtransferases/genética , Sulfurtransferases/metabolismo
7.
Nature ; 568(7753): 571-575, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944476

RESUMO

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.


Assuntos
ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Ciclo do Ácido Cítrico , Evolução Molecular , ATP Citrato (pro-S)-Liase/genética , Biocatálise , Chlorobium/enzimologia , Chlorobium/genética , Cristalografia por Raios X , Humanos , Methanosarcinales/enzimologia , Methanosarcinales/genética , Modelos Moleculares
8.
Med Hypotheses ; 123: 72-73, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30696597

RESUMO

The paper published by Ruczyszky and Liu (2017) reports on the biosynthesis of ergothioneine under both aerobic and anaerobic conditions. We would like to suggest a hypothesis as to what could be the reason that microorganisms on the Earth synthesized ergothioneine under anaerobic conditions.


Assuntos
Atmosfera , Planeta Terra , Ergotioneína/química , Oxigênio , Antioxidantes , Bactérias/metabolismo , Catálise , Chlorobium/metabolismo , Elétrons , Histidina/química
9.
Biochem J ; 476(1): 51-66, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30538153

RESUMO

The LRR (leucine-rich repeat)-Roc (Ras of complex proteins)-COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer-monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5'-diphosphate)-bound states and monomeric in the GTP (guanosine-5'-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR-Roc-COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the 'Roc dimerization loop'. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.


Assuntos
Proteínas de Bactérias/química , Chlorobium/enzimologia , Dimerização , Guanosina Trifosfato/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Simulação de Dinâmica Molecular , Proteínas de Bactérias/genética , Chlorobium/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Estrutura Terciária de Proteína
10.
J Biol Chem ; 293(39): 15233-15242, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30126840

RESUMO

Chlorobaculum tepidum, a green sulfur bacterium, utilizes chlorobactene as its major carotenoid, and this organism also accumulates a reduced form of this monocyclic pigment, 1',2'-dihydrochlorobactene. The protein catalyzing this reduction is the last unidentified enzyme in the biosynthetic pathways for all of the green sulfur bacterial pigments used for photosynthesis. The genome of C. tepidum contains two paralogous genes encoding members of the FixC family of flavoproteins: bchP, which has been shown to encode an enzyme of bacteriochlorophyll biosynthesis; and bchO, for which a function has not been assigned. Here we demonstrate that a bchO mutant is unable to synthesize 1',2'-dihydrochlorobactene, and when bchO is heterologously expressed in a neurosporene-producing mutant of the purple bacterium, Rhodobacter sphaeroides, the encoded protein is able to catalyze the formation of 1,2-dihydroneurosporene, the major carotenoid of the only other organism reported to synthesize 1,2-dihydrocarotenoids, Blastochloris viridis Identification of this enzyme completes the pathways for the synthesis of photosynthetic pigments in Chlorobiaceae, and accordingly and consistent with its role in carotenoid biosynthesis, we propose to rename the gene cruI Notably, the absence of cruI in B. viridis indicates that a second 1,2-carotenoid reductase, which is structurally unrelated to CruI (BchO), must exist in nature. The evolution of this carotenoid reductase in green sulfur bacteria is discussed herein.


Assuntos
Bacterioclorofilas/biossíntese , Carotenoides/biossíntese , Chlorobi/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacterioclorofilas/química , Bacterioclorofilas/genética , Vias Biossintéticas/genética , Carotenoides/química , Carotenoides/genética , Carotenoides/metabolismo , Chlorobi/química , Chlorobium/enzimologia , Chlorobium/genética , Genoma Bacteriano/genética , Oxirredutases/química , Oxirredutases/genética , Fotossíntese/genética
11.
J Phys Chem B ; 122(14): 3734-3743, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29554425

RESUMO

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub821 and Sub817) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Chlorobium/química , Chlorobium/genética , Transferência de Energia , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Espectrometria de Fluorescência , Fenilalanina , Fotossíntese , Tirosina
12.
Environ Microbiol Rep ; 10(2): 179-183, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29393582

RESUMO

Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N2 and CO2 fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer-scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H2 S (i.e. electron donor) limited chemostats show that varying levels of NH4+ limitation induce heterogeneity in N2 fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H2 S) populations indicates that electron donor limitation drives heterogeneity in N2 and CO2 fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.


Assuntos
Chlorobium/metabolismo , Sulfeto de Hidrogênio/metabolismo , Enxofre/metabolismo , Chlorobium/classificação , Chlorobium/genética , Chlorobium/isolamento & purificação , Transporte de Elétrons , Fixação de Nitrogênio , Fenótipo , Espectrometria de Massa de Íon Secundário
13.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 11): 629-634, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29095157

RESUMO

The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of L-threo-tetrahydrobiopterin to 1-O-(L-threo-biopterin-2'-yl)-ß-N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24 M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15 Šresolution from a single CtPGT crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 189.61, b = 79.98, c = 105.92 Å, ß = 120.5°.


Assuntos
Chlorobium/enzimologia , Glicosiltransferases/química , Pteridinas/metabolismo , Cristalização , Cristalografia por Raios X , Glicosiltransferases/metabolismo , Conformação Proteica
15.
Nat Commun ; 8(1): 1008, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29044096

RESUMO

Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chlorobium/enzimologia , Guanosina Trifosfato/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/enzimologia , Proteínas de Bactérias/genética , Chlorobium/química , Chlorobium/genética , Dimerização , Humanos , Hidrólise , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Doença de Parkinson/genética , Fosforilação , Estrutura Terciária de Proteína
16.
Geobiology ; 15(6): 784-797, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29035021

RESUMO

Little Salt Spring (Sarasota County, FL, USA) is a sinkhole with groundwater vents at ~77 m depth. The entire water column experiences sulfidic (~50 µM) conditions seasonally, resulting in a system poised between oxic and sulfidic conditions. Red pinnacle mats occupy the sediment-water interface in the sunlit upper basin of the sinkhole, and yielded 16S rRNA gene clones affiliated with Cyanobacteria, Chlorobi, and sulfate-reducing clades of Deltaproteobacteria. Nine bacteriochlorophyll e homologues and isorenieratene indicate contributions from Chlorobi, and abundant chlorophyll a and pheophytin a are consistent with the presence of Cyanobacteria. The red pinnacle mat contains hopanoids, including 2-methyl structures that have been interpreted as biomarkers for Cyanobacteria. A single sequence of hpnP, the gene required for methylation of hopanoids at the C-2 position, was recovered in both DNA and cDNA libraries from the red pinnacle mat. The hpnP sequence was most closely related to cyanobacterial hpnP sequences, implying that Cyanobacteria are a source of 2-methyl hopanoids present in the mat. The mats are capable of light-dependent primary productivity as evidenced by 13 C-bicarbonate photoassimilation. We also observed 13 C-bicarbonate photoassimilation in the presence of DCMU, an inhibitor of electron transfer to Photosystem II. Our results indicate that the mats carry out light-driven primary production in the absence of oxygen production-a mechanism that may have delayed the oxygenation of the Earth's oceans and atmosphere during the Proterozoic Eon. Furthermore, our observations of the production of 2-methyl hopanoids by Cyanobacteria under conditions of low oxygen and low light are consistent with the recovery of these structures from ancient black shales as well as their paucity in modern marine environments.


Assuntos
Processos Autotróficos , Chlorobium/metabolismo , Cianobactérias/metabolismo , Água Subterrânea/microbiologia , Processos Fototróficos , Aerobiose , Anaerobiose , Biomarcadores/análise , Florida , Paleontologia
17.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 9): 520-524, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28876231

RESUMO

Roco proteins are characterized by the presence of a Roc-COR supradomain harbouring GTPase activity, which is often preceded by an LRR domain. The most notorious member of the Roco protein family is the Parkinson's disease-associated LRRK2. The Roco protein from the bacterium Chlorobium tepidum has been used as a model system to investigate the structure and mechanism of this class of enzymes. Here, the crystallization and crystallographic analysis of the LRR-Roc-COR construct of the C. tepidum Roco protein is reported. The LRR-Roc-COR crystals belonged to space group P212121, with unit-cell parameters a = 95.6, b = 129.8, c = 179.5 Å, α = ß = γ = 90°, and diffracted to a resolution of 3.3 Å. Based on the calculated Matthews coefficient, Patterson map analysis and an initial molecular-replacement analysis, one protein dimer is present in the asymmetric unit. The crystal structure of this protein will provide valuable insights into the interaction between the Roc-COR and LRR domains within Roco proteins.


Assuntos
Proteínas de Bactérias/química , Chlorobium/enzimologia , Cristalização/métodos , GTP Fosfo-Hidrolases/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
18.
Angew Chem Int Ed Engl ; 56(41): 12508-12511, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28786519

RESUMO

Ergothioneine is a sulfur metabolite that occurs in microorganisms, fungi, plants, and animals. The physiological function of ergothioneine is not clear. In recent years broad scientific consensus has formed around the idea that cellular ergothioneine primarily protects against reactive oxygen species. Herein we provide evidence that this focus on oxygen chemistry may be too narrow. We describe two enzymes from the strictly anaerobic green sulfur bacterium Chlorobium limicola that mediate oxygen-independent biosynthesis of ergothioneine. This anoxic origin suggests that ergothioneine is also important for oxygen-independent life. Furthermore, one of the discovered ergothioneine biosynthetic enzymes provides the first example of a rhodanese-like enzyme that transfers sulfur to non-activated carbon.


Assuntos
Proteínas de Bactérias/metabolismo , Chlorobium/metabolismo , Ergotioneína/metabolismo , Anaerobiose , Vias Biossintéticas , Chlorobium/enzimologia , Oxigênio/metabolismo
19.
FEMS Microbiol Ecol ; 93(5)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28431154

RESUMO

Anoxygenic photoautotrophic bacteria which use light energy and electrons from Fe(II) for growth, so-called photoferrotrophs, are suggested to have been amongst the first phototrophic microorganisms on Earth and to have contributed to the deposition of sedimentary iron mineral deposits, i.e. banded iron formations. To date only two isolates of marine photoferrotrophic bacteria exist, both of which are closely related purple non-sulfur bacteria. Here we present a novel green-sulfur photoautotrophic Fe(II) oxidizer isolated from a marine coastal sediment, Chlorobium sp. strain N1, which is closely related to the freshwater green-sulfur bacterium Chlorobium luteolum DSM273 that is incapable of Fe(II) oxidation. Besides Fe(II), our isolated strain grew phototrophically with other inorganic and organic substrates such as sulfide, hydrogen, lactate or yeast extract. Highest Fe(II) oxidation rates were measured at pH 7.0-7.3, the temperature optimum was 25°C. Mössbauer spectroscopy identified ferrihydrite as the main Fe(III) mineral and fluorescence and helium-ion microscopy revealed cell-mineral aggregates without obvious cell encrustation. In summary, our study showed that the new isolate is physiologically adapted to the conditions of its natural habitat but also to conditions as proposed for early Earth and is thus a suitable model organism for further studies addressing phototrophic Fe(II) oxidation on early Earth.


Assuntos
Chlorobium , Compostos Férricos/metabolismo , Sedimentos Geológicos/microbiologia , Chlorobium/classificação , Chlorobium/isolamento & purificação , Chlorobium/metabolismo , Compostos Ferrosos/metabolismo , Água Doce/microbiologia , Ferro/metabolismo , Luz , Oxirredução , Enxofre/metabolismo , Temperatura
20.
Glycobiology ; 27(5): 469-476, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28104786

RESUMO

Glycosaminoglycans (GAGs) are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria Pasteurella multocida Type F and Escherichia coli K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, Chlorobium phaeobacteroides DSM 266, which was very similar (~62% identical) to the open reading frames of the known bifunctional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant E. coli-derived Chlorobium enzyme preparations were found to possess bona fide chondroitin synthase activity in vitro. This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in C. phaeobacteroides is unexpected for several reasons including (a) a free-living nonpathogenic organism should not "need" an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5-3 billion years ago and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.


Assuntos
Chlorobium/enzimologia , Glicosaminoglicanos/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/isolamento & purificação , Sequência de Aminoácidos/genética , Sulfatos de Condroitina/química , Escherichia coli/genética , Glicosaminoglicanos/genética , N-Acetilgalactosaminiltransferases/metabolismo , Especificidade por Substrato
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