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1.
J Agric Food Chem ; 69(20): 5755-5763, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33988022

RESUMO

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 Å (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (ΔCBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the "lid model") to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Rhodothermus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos , Rhodothermus/metabolismo , Especificidade por Substrato
2.
Protein J ; 40(2): 184-191, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33651244

RESUMO

Substrate binding proteins (SBPs) bind to specific ligands in the periplasmic regions of cells and then bind to membrane proteins to participate in transport or signal transduction. Typically, SBPs consist of two α/ß domains and recognize the substrate by a flexible hinge region between the two domains. Conversely, the short-length SBPs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes. We previously determined the crystal structure of Rhodothermus marinus SBP (named as RmSBP), consisting of a single α/ß domain; however, the substrate recognition mechanism is still unclear. To better understand the functions of short length RmSBP, we performed a comprehensive study, involving comparative structure analysis, computational substrate docking, and X-ray crystallographic data. RmSBP shares a high level of similarity in the α/ß domain region with other SBPs, but it has a distinct topology in the C-terminal domain. The substrate binding model suggested that conformational changes in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility in the ß1-α2 loop, ß5-ß6 loop, and extended C-terminal domains, based on the electron density map and temperature B-factor analysis. These results provide information that will further our understanding on the functions of the short length SBP.


Assuntos
Proteínas de Bactérias/metabolismo , Rhodothermus/metabolismo , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica
3.
Protein Expr Purif ; 164: 105478, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31421223

RESUMO

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 °C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Proteínas de Bactérias/genética , Rhodothermus/genética , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodothermus/química , Rhodothermus/metabolismo
4.
Protein Expr Purif ; 164: 105464, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31376486

RESUMO

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.


Assuntos
Proteínas de Bactérias/genética , Endo-1,4-beta-Xilanases/genética , Rhodothermus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhodothermus/química , Rhodothermus/isolamento & purificação , Rhodothermus/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
5.
Metab Eng ; 44: 182-190, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29037779

RESUMO

Thermophilic organisms are being increasingly investigated and applied in metabolic engineering and biotechnology. The distinct metabolic and physiological characteristics of thermophiles, including broad substrate range and high uptake rates, coupled with recent advances in genetic tool development, present unique opportunities for strain engineering. However, poor understanding of the cellular physiology and metabolism of thermophiles has limited the application of systems biology and metabolic engineering tools to these organisms. To address this concern, we applied high resolution 13C metabolic flux analysis to quantify fluxes for three divergent extremely thermophilic bacteria from separate phyla: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252. We performed 18 parallel labeling experiments, using all singly labeled glucose tracers for each strain, reconstructed and validated metabolic network models, measured biomass composition, and quantified precise metabolic fluxes for each organism. In the process, we resolved many uncertainties regarding gaps in pathway reconstructions and elucidated how these organisms maintain redox balance and generate energy. Overall, we found that the metabolisms of the three thermophiles were highly distinct, suggesting that adaptation to growth at high temperatures did not favor any particular set of metabolic pathways. All three strains relied heavily on glycolysis and TCA cycle to generate key cellular precursors and cofactors. None of the investigated organisms utilized the Entner-Doudoroff pathway and only one strain had an active oxidative pentose phosphate pathway. Taken together, the results from this study provide a solid foundation for future model building and engineering efforts with these and related thermophiles.


Assuntos
Isótopos de Carbono/metabolismo , Geobacillus/metabolismo , Temperatura Alta , Metaboloma , Modelos Biológicos , Rhodothermus/metabolismo , Thermus thermophilus/metabolismo
6.
Carbohydr Polym ; 156: 1-8, 2017 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-27842803

RESUMO

The thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content.


Assuntos
Polissacarídeos Bacterianos/biossíntese , Rhodothermus/metabolismo , Amino Açúcares/química , Arabinose/química , Glucose/metabolismo , Lactose/metabolismo , Maltose/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/isolamento & purificação , Rhodothermus/química , Rhodothermus/classificação , Sacarose/metabolismo , Ácidos Urônicos/química , Xilose/química
7.
Extremophiles ; 21(2): 283-296, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27928680

RESUMO

Recent studies have revealed the physiological significance of post-translational lysine acylations such as acetylation in the regulation of various cellular processes. Here, we characterized lysine propionylation, a recently discovered post-translational acylation, in five representative bacteria: Geobacillus kaustophilus, Thermus thermophilus, Escherichia coli, Bacillus subtilis, and Rhodothermus marinus. Using antibody-based propionyl peptide enrichment followed by identification with nano-liquid chromatography tandem mass spectrometry, we showed that proteins were subject to lysine propionylation in all five bacterial species analyzed. Notably, many propionylations were identified in the Bacillus-related, thermophilic eubacterium G. kaustophilus, but fewer in the mesophilic eubacterium B. subtilis, suggesting that propionylation event abundance is independent of phylogenetic relationship. We further found propionylation sites in the thermophilic eubacterium T. thermophilus, but the thermophilic eubacterium R. marinus showed the fewest number of sites, indicating that growth temperature is not a determinant of propionylation state. In silico analyses demonstrated that lysine propionylation is related to metabolic pathways, particularly those controlled by acyl-CoA synthetases, similar to lysine acetylation. We also detected dozens of propionylation sites at positions important for protein functions across bacteria, demonstrating the regulatory mechanisms affected by lysine propionylations. Our proteome-wide analyses across bacteria thus provide insights into the general functions of lysine propionylation.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Geobacillus/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Rhodothermus/metabolismo , Thermus thermophilus/metabolismo , Acetilação , Lisina/metabolismo , Propionatos/metabolismo , Proteômica
8.
Biochim Biophys Acta Proteins Proteom ; 1865(2): 232-242, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27888076

RESUMO

Lysine succinylation, one of post-translational acylations conserved from eukaryotes to bacteria, plays regulatory roles in various cellular processes. However, much remains unknown about the general and specific characteristics of lysine succinylation among bacteria, and about its functions different from those of other acylations. In this study, we characterized lysine succinylation, a newly discovered widespread type of lysine acylation in five bacterial species with different characteristics such as optimal growth temperature and cell wall structure. This study is the first to demonstrate that succinylation is general phenomenon occurring not only in mesophiles but also in thermophiles. Mapping of succinylation sites on protein structures revealed that succinylation occurs at many lysine residues important for protein function. Comparison of the succinylation sites in the five bacterial species provides insights regarding common protein regulation mechanisms utilizing lysine succinylation. Many succinylation sites were conserved among five bacteria, especially between Geobacillus kaustophilus and Bacillus subtilis, some of which are functionally important sites. Furthermore, systematic comparison of the succinyl-proteome results and our previous propionyl-proteome results showed that the abundance of these two types of acylations is considerably different among the five bacteria investigated. Many succinylation and propionylation events were detected in G. kaustophilus, whereas Escherichia coli and B. subtilis exhibited high succinylation and low propionylation; low succinylation and high propionylation were identified in Thermus thermophilus, and low succinylation and propionylation were observed in Rhodothermus marinus. Comparison of the characteristics of lysine succinylation and lysine propionylation suggested these two types of acylation play different roles in cellular processes.


Assuntos
Acilação/fisiologia , Lisina/metabolismo , Proteoma/metabolismo , Ácido Succínico/metabolismo , Thermus thermophilus/metabolismo , Acetilação , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Geobacillus/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Rhodothermus/metabolismo
9.
Structure ; 24(2): 243-51, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26749448

RESUMO

The ß-barrel assembly machine (BAM) mediates folding and insertion of integral ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermus marinus. The POTRA5 domain binds BamD between its tetratricopeptide repeats 3 and 4. The interface structural elements are conserved in the Escherichia coli proteins, which allowed structure validation by mutagenesis and disulfide crosslinking in E. coli. Furthermore, the interface is consistent with previously reported mutations that impair BamA-BamD binding. The structure serves as a linchpin to generate a BAM model where POTRA domains and BamD form an elongated periplasmic ring adjacent to the membrane with a central cavity approximately 30 × 60 Å wide. We propose that nascent OMPs bind this periplasmic ring prior to insertion and folding by BAM.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Rhodothermus/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Modelos Moleculares , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rhodothermus/química , Rhodothermus/genética
10.
Appl Microbiol Biotechnol ; 99(22): 9463-72, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26033773

RESUMO

UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn(2+) ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.


Assuntos
Vias Biossintéticas , Rhodothermus/metabolismo , Uridina Difosfato Xilose/biossíntese , Biocatálise , Carboxiliases/genética , Carboxiliases/metabolismo , Clonagem Molecular , Fontes Termais/microbiologia , Cinética , Proteínas Recombinantes/metabolismo , Rhodothermus/enzimologia , Rhodothermus/genética , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Ácido Glucurônico/biossíntese , Uridina Difosfato Ácido Glucurônico/genética , Uridina Difosfato Ácido Glucurônico/metabolismo , Xilose/biossíntese , Xilose/metabolismo
11.
Environ Microbiol ; 17(7): 2492-504, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25472423

RESUMO

We describe a novel biosynthetic pathway for glycerophosphoinositides in Rhodothermus marinus in which inositol is activated by cytidine triphosphate (CTP); this is unlike all known pathways that involve activation of the lipid group instead. This work was motivated by the detection in the R. marinus genome of a gene with high similarity to CTP:L-myo-inositol-1-phosphate cytidylyltransferase, the enzyme that synthesizes cytidine diphosphate (CDP)-inositol, a metabolite only known in the synthesis of di-myo-inositol phosphate. However, this solute is absent in R. marinus. The fate of radiolabelled CDP-inositol was investigated in cell extracts to reveal that radioactive inositol was incorporated into the chloroform-soluble fraction. Mass spectrometry showed that the major lipid product has a molecular mass of 810 Da and contains inositol phosphate and alkyl chains attached to glycerol by ether bonds. The occurrence of ether-linked lipids is rare in bacteria and has not been described previously in R. marinus. The relevant synthase was identified by functional expression of the candidate gene in Escherichia coli. The enzyme catalyses the transfer of L-myo-inositol-1-phosphate from CDP-inositol to dialkylether glycerol yielding dialkylether glycerophosphoinositol. Database searching showed homologous proteins in two bacterial classes, Sphingobacteria and Alphaproteobacteria. This is the first report of the involvement of CDP-inositol in phospholipid synthesis.


Assuntos
Cistina Difosfato/metabolismo , Citidina Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Inositol/metabolismo , Fosfatidilinositóis/biossíntese , Rhodothermus/metabolismo , Vias Biossintéticas , Nucleotidiltransferases/metabolismo
12.
BMC Biotechnol ; 14: 113, 2014 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-25540113

RESUMO

BACKGROUND: Derivatized celluloses, such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC), are of pharmaceutical importance and extensively employed in tablet matrices. Each batch of derivatized cellulose is thoroughly characterized before utilized in tablet formulations as batch-to-batch differences can affect drug release. The substitution pattern of the derivatized cellulose polymers, i.e. the mode on which the substituent groups are dispersed along the cellulose backbone, can vary from batch-to-batch and is a factor that can influence drug release. RESULTS: In the present study an analytical approach for the characterization of the substitution pattern of derivatized celluloses is presented, which is based on the use of carbohydrate-binding modules (CBMs) and affinity electrophoresis. CBM4-2 from Rhodothermus marinus xylanase 10A is capable of distinguishing between batches of derivatized cellulose with different substitution patterns. This is demonstrated by a higher migration retardation of the CBM in acrylamide gels containing batches of MC and HPMC with a more heterogeneous distribution pattern. CONCLUSIONS: We conclude that CBMs have the potential to characterize the substitution pattern of cellulose derivatives and anticipate that with use of CBMs with a very selective recognition capacity it will be possible to more extensively characterize and standardize important carbohydrates used for instance in tablet formulation.


Assuntos
Proteínas de Bactérias/química , Celulose/química , Receptores de Superfície Celular/química , Rhodothermus/metabolismo , Proteínas de Bactérias/metabolismo , Estrutura Molecular , Receptores de Superfície Celular/metabolismo , Rhodothermus/química
13.
PLoS One ; 7(5): e37010, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22649505

RESUMO

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.


Assuntos
Biocombustíveis , Biotecnologia/métodos , Celulases/metabolismo , Líquidos Iônicos/metabolismo , Lignina/metabolismo , Panicum/química , Escherichia coli/metabolismo , Glicosídeo Hidrolases , Paenibacillus/genética , Paenibacillus/metabolismo , Proteômica , Rhodothermus/genética , Rhodothermus/metabolismo , Análise de Sequência de DNA , Temperatura , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
14.
J Mol Biol ; 409(3): 348-57, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21463635

RESUMO

Folding and insertion of integral ß-barrel proteins in the outer membrane (OM) is an essential process for Gram-negative bacteria that requires the ß-barrel assembly machinery (BAM). Efficient OM protein (OMP) folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the OM and, in Escherichia coli, consists of the ß-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria, whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability, suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here, we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains five tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining two TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Rhodothermus/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
15.
ACS Chem Biol ; 6(5): 477-83, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21268658

RESUMO

The energy transduction by complex I from Rhodothermusmarinus was addressed by studying the influence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the activities of this enzyme. EIPA is an inhibitor of both Na(+)/H(+) antiporter and complex I NADH:quinone oxidoreductase activity. We performed studies of NADH:quinone oxidoreductase and H(+) and Na(+) translocation activities of complex I from R. marinus at different concentrations of EIPA, using inside-out membrane vesicles. We observed that the oxidoreductase activity and both H(+) and Na(+) transports are inhibited by EIPA. Most interestingly, the catalytic and the two transport activities showed different inhibition profiles. The transports are inhibited at concentrations of EIPA at which the catalytic activity is not affected. In this way the catalytic and transport activities were decoupled. Moreover, the inhibition of the catalytic activity was not influenced by the presence of Na(+), whereas the transport of H(+) showed different inhibition behaviors in the presence and absence of Na(+). Taken together our observations indicate that complex I from R. marinus performs energy transduction by two different processes: proton pumping and Na(+)/H(+) antiporting. The decoupling of the catalytic and transport activities suggests the involvement of an indirect coupling mechanism, possibly through conformational changes.


Assuntos
Amilorida/análogos & derivados , Complexo I de Transporte de Elétrons/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Amilorida/farmacologia , Transporte Biológico/efeitos dos fármacos , Catálise , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Quinona Redutases/antagonistas & inibidores , Rhodothermus/metabolismo , Sódio/metabolismo , Valinomicina/farmacologia
16.
Biochim Biophys Acta ; 1807(3): 286-92, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21172303

RESUMO

The nature of the ions that are translocated by Escherichia coli and Paracoccus denitrificans complexes I was investigated. We observed that E. coli complex I was capable of proton translocation in the same direction to the established deltapsi, showing that in the tested conditions, the coupling ion is the H(+). Furthermore, Na(+) transport to the opposite direction was also observed, and, although Na(+) was not necessary for the catalytic or proton transport activities, its presence increased the latter. We also observed H(+) translocation by P. denitrificans complex I, but in this case, H(+) transport was not influenced by Na(+) and also Na(+) transport was not observed. We concluded that E. coli complex I has two energy coupling sites (one Na(+) independent and the other Na(+) dependent), as previously observed for Rhodothermus marinus complex I, whereas the coupling mechanism of P. denitrificans enzyme is completely Na(+) independent. This work thus shows that complex I energy transduction by proton pumping and Na(+)/H(+) antiporting is not exclusive of the R. marinus enzyme. Nevertheless, the Na(+)/H(+) antiport activity seems not to be a general property of complex I, which may be correlated with the metabolic characteristics of the organisms.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Escherichia coli/metabolismo , NAD/metabolismo , Paracoccus denitrificans/metabolismo , Sódio/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Transporte de Íons , Cinética , Espectroscopia de Ressonância Magnética , Potenciais da Membrana/efeitos dos fármacos , Oxirredutases/metabolismo , Consumo de Oxigênio , Paracoccus denitrificans/efeitos dos fármacos , Prótons , Rhodothermus/metabolismo
17.
Biochim Biophys Acta ; 1797(8): 1477-82, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20206595

RESUMO

An alternative complex III (ACIII) is a respiratory complex with quinol:electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc1 complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa3 oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidences, taken together with mass spectrometry revealed a structural association between ACIII and caa3 oxygen reductase. The association was also shown to be functional, since quinol:oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.


Assuntos
Grupo dos Citocromos c/química , Citocromos a3/química , Citocromos a/química , Complexo III da Cadeia de Transporte de Elétrons/química , Rhodothermus/metabolismo , Grupo dos Citocromos c/fisiologia , Citocromos a/fisiologia , Citocromos a3/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/fisiologia , Fluorescência , Família Multigênica , Vitamina K/análogos & derivados , Vitamina K/química
18.
Biochim Biophys Acta ; 1797(4): 509-15, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20100453

RESUMO

A sodium ion efflux, together with a proton influx and an inside-positive DeltaPsi, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by (23)Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.


Assuntos
Proteínas de Bactérias/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Transferência de Energia , Rhodothermus/metabolismo , Membrana Celular/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Transporte de Íons/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , NAD/metabolismo , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Oxigênio/metabolismo , Consumo de Oxigênio , Cianeto de Potássio/farmacologia , Força Próton-Motriz , Prótons , Rotenona/farmacologia , Sódio/metabolismo , Espectrometria de Fluorescência
19.
Biochim Biophys Acta ; 1784(11): 1771-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18555809

RESUMO

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.


Assuntos
Celulases/química , Engenharia de Proteínas/métodos , Rhodothermus/enzimologia , Homologia Estrutural de Proteína , Temperatura , Trichoderma/enzimologia , Sequência de Aminoácidos , Domínio Catalítico/genética , Celulases/genética , Celulases/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Desnaturação Proteica/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , Rhodothermus/genética , Rhodothermus/metabolismo , Propriedades de Superfície , Termodinâmica , Temperatura de Transição , Trichoderma/genética , Trichoderma/metabolismo
20.
J Bacteriol ; 190(6): 1871-8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18192391

RESUMO

Trehalose uptake at 65 degrees C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: Km = 156 +/- 11 microM and Vmax = 21.2 +/- 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (Km = 0.11 +/- 0.03 microM and Vmax = 0.39 +/- 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (Km = 46 +/- 3 microM and Vmax = 48 +/- 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 microM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.


Assuntos
Proteínas de Bactérias/fisiologia , Rhodothermus/metabolismo , Trealase/metabolismo , Trealose/metabolismo , Arseniatos/farmacologia , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Etanol/farmacologia , Glucose/metabolismo , Glucose/farmacocinética , Isomaltose/análogos & derivados , Isomaltose/metabolismo , Isomaltose/farmacocinética , Cinética , Modelos Teóricos , Periplasma/enzimologia , Rhodothermus/efeitos dos fármacos , Fluoreto de Sódio/farmacologia , Trealose/farmacocinética
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