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1.
Nat Commun ; 11(1): 769, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034139

RESUMO

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.


Assuntos
Histidina Quinase/química , Histidina Quinase/metabolismo , Thermotoga maritima/enzimologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Histidina Quinase/genética , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação , Conformação Proteica , Thermotoga maritima/genética , Transativadores/química , Transativadores/metabolismo
2.
Biochim Biophys Acta Bioenerg ; 1861(1): 148087, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669490

RESUMO

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxinred- + NAD(P)H + 3 H+ = 2 ferredoxinox + NAD(P)+ + 2 H2). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH2 higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (~10 kJ mol-1 H2) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E°' largely determines the sensitivity of the bifurcating reaction to pH2, modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the 'Thauer limit' (4 H2 per glucose) as a function of temperature and pH2. For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 °C if pH 2 ≤ ~10 mbar. The results also predict Archaea, expressing a non-typical glycolysis pathway, would not benefit from a bifurcating [FeFe]-hydrogenase reaction; interestingly, no Archaea have been observed experimentally with a [FeFe]-hydrogenase enzyme.


Assuntos
Proteínas de Bactérias , Hidrogênio , Hidrogenase , Proteínas com Ferro-Enxofre , Thermotoga maritima/enzimologia , Anaerobiose/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrogênio/química , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Oxirredução , Termodinâmica
3.
Extremophiles ; 23(6): 659-667, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31338597

RESUMO

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) overexpression, attempting to provide excess EPSPS to combine with glyphosate, is one way to improve glyphosate resistance of plants. The EPSPS in extremophiles which is selected by nature to withstand the evolutionary pressure may possess some potential-specific biological functions. In this study, we reported the cloning, expression and enzymatic characterization of a novel Class II EPSPS AroAT. maritima from Thermotoga maritima MSB8. The enzyme showed low sequence identities with other EPSPSs, and was one of the most thermostable EPSPSs so far, which showed the optimum enzyme activity at 80 °C. The enzyme maintains the activity below 50 °C and in a wide range of pH 4.0-10, which indicated its stability under rough environment, especially in tropical regions and alkaline soil. Excellent Ki/Km value of AroAT. maritima suggested that the enzyme showed powerful competitive binding capacity of PEP over glyphosate and high glyphosate tolerance. Furthermore, aroAT. maritima gene was transformed into Arabidopsis thaliana. The transgenic lines were resistant to 15 mM glyphosate, which proved the application value in the cultivation of glyphosate-tolerant plants.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase , Arabidopsis , Proteínas de Bactérias , Farmacorresistência Bacteriana/genética , Escherichia coli , Glicina/análogos & derivados , Microrganismos Geneticamente Modificados , Plantas Geneticamente Modificadas , Thermotoga maritima , 3-Fosfoshikimato 1-Carboxiviniltransferase/biossíntese , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Glicina/farmacologia , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Thermotoga maritima/enzimologia , Thermotoga maritima/genética
4.
Appl Microbiol Biotechnol ; 103(15): 6141-6151, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31190240

RESUMO

AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary structural comparison suggest that AmyC could actually be a glycogen-branching enzyme (GBE), a key enzyme in the biosynthesis of glycogen. This communication presents phylogenetic and biochemical evidence that AmyC is a GBE with a relatively high hydrolytic (α-amylase) activity (up to 30% of the total activity), creating a branched α-glucan with 8.5% α-1,6-glycosidic bonds. The high hydrolytic activity is explained by the fact that AmyC has a considerably shorter catalytic loop (residues 213-220) not reaching the acceptor side. Secondly, in AmyC, the tryptophan residue (W 246) near the active site has its side chain buried in the protein interior, while the side chain is at the surface in Tk1436 and Tt1467 GBEs. The putative GBEs from three other Thermotogaceae, with very high sequence similarities to AmyC, were found to have the same structural elements as AmyC, suggesting that GH57 GBEs with relatively high hydrolytic activity may be widespread in nature.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Thermotoga maritima/enzimologia , alfa-Amilases/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Hidrólise , Modelos Moleculares , Filogenia , Conformação Proteica , Homologia de Sequência de Aminoácidos , alfa-Amilases/genética
5.
Nucleic Acids Res ; 47(8): 4136-4152, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30892613

RESUMO

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/química , Reparo do DNA , DNA Bacteriano/química , Endodesoxirribonucleases/química , Proteínas de Escherichia coli/química , Geobacillus stearothermophilus/enzimologia , ortoaminobenzoatos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Dano ao DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Cinética , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Thermotoga maritima/química , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , ortoaminobenzoatos/metabolismo
6.
Enzyme Microb Technol ; 125: 53-62, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885325

RESUMO

The thermostable ß-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short-chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70 °C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1-kestotriose, 6G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6-kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3-fold higher than that of the wild-type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg-1, represents an attractive candidate for the continuous production of FOS-containing invert syrup at pasteurization temperatures.


Assuntos
Proteínas de Bactérias/metabolismo , Oligossacarídeos/biossíntese , Thermotoga maritima/enzimologia , beta-Frutofuranosidase/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Oligossacarídeos/química , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sacarose/metabolismo , Temperatura , Thermotoga maritima/genética , beta-Frutofuranosidase/genética
7.
J Chem Theory Comput ; 15(4): 2116-2126, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30836000

RESUMO

To understand how protein function changes upon an allosteric perturbation, such as ligand binding and mutation, significant progress in characterizing allosteric network from molecular dynamics (MD) simulations has been made. However, determining which amino acid(s) play an essential role in the propagation of signals may prove challenging, even when the location of the source and sink is known for a protein or protein complex. This challenge is mainly due to the large fluctuations in protein dynamics that cause instability of the network topology within a single trajectory or between multiple replicas. To solve this problem, we introduce the current-flow betweenness scheme, originated from electrical network theory, to protein dynamical network analysis. To demonstrate the benefit of this new method, we chose a prototypic allosteric enzyme (IGPS or HisH-HisF dimer) as our benchmark system. Using multiple replicas of simulations and multiple network topology comparison metrics (edge ranking, path length, and node frequency), we show that the current-flow betweenness provides a significant improvement in the convergence of the allosteric networks. The improved stability of the network topology allows us to generate a delta-network between the apo and holo forms of the protein. We illustrated that the delta-network is a more rigorous way to capture the subtle changes in the networks that would otherwise be neglected by comparing node usage frequencies alone. We have also investigated the use of a linear smoothing function to improve the stability of the contact map. The methodology presented here is general and may be applied to other topology and weighting schemes. We thus conclude that, for determining protein signaling pathways between the pair(s) of source and sink, multiple MD simulation replicas are necessary, and the current-flow betweenness scheme introduced here provides a more robust approach than the geodesic scheme based on correlation edge weighting.


Assuntos
Aminoidrolases/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Thermotoga maritima/enzimologia , Regulação Alostérica , Aminoidrolases/química , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Thermotoga maritima/química , Thermotoga maritima/metabolismo
8.
Molecules ; 24(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866470

RESUMO

The two-component system (TCS) is a significant signal transduction system for bacteria to adapt to complicated and variable environments, and thus has recently been regarded as a novel target for developing antibacterial agents. The natural product luteolin (Lut) can inhibit the autophosphorylation activity of the typical histidine kinase (HK) HK853 from Thermotoga maritime, but the inhibition mechanism is not known. Herein, we report on the binding mechanism of a typical flavone with HK853 by using solution NMR spectroscopy, isothermal titration calorimetry (ITC), and molecular docking. We show that luteolin inhibits the activity of HK853 by occupying the binding pocket of adenosine diphosphate (ADP) through hydrogen bonds and π-π stacking interaction structurally. Our results reveal a detailed mechanism for the inhibition of flavones and observe the conformational and dynamics changes of HK. These results should provide a feasible approach for antibacterial agent design from the view of the histidine kinases.


Assuntos
Histidina Quinase/antagonistas & inibidores , Histidina Quinase/química , Luteolina/farmacologia , Thermotoga maritima/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Calorimetria , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Thermotoga maritima/química
9.
Metab Eng ; 52: 1-8, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30389613

RESUMO

The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20 mM xylose to 16.1 mM inositol with a conversion efficiency of 96.6% at 70 °C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70 °C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.


Assuntos
Suplementos Nutricionais/análise , Engenharia Metabólica/métodos , Açúcares/química , Madeira/química , Xilose/química , Trifosfato de Adenosina/metabolismo , Archaeoglobus/enzimologia , Archaeoglobus/metabolismo , Catálise , Inositol/metabolismo , Magnésio/metabolismo , Redes e Vias Metabólicas , NAD/metabolismo , Fosforilação , Thermotoga maritima/enzimologia , Thermus/enzimologia , Thermus/metabolismo
10.
FEBS J ; 286(3): 601-614, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30548096

RESUMO

Various d-amino acids are involved in peptidoglycan and biofilm metabolism in bacteria, suggesting that these compounds are necessary for successful adaptation to environmental changes. In addition to the conventional d-alanine (d-Ala) and d-glutamate, the peptidoglycan of the hyperthermophilic bacterium Thermotoga maritima contains both l-lysine (l-Lys) and d-Lys, but not meso-diaminopimelate (meso-Dpm). d-Lys is an uncommon component of peptidoglycan, and its biosynthetic pathway remains unclear. In this study, we identified and characterized a novel Lys racemase (TM1597) and Dpm epimerase (TM1522) associated with the d-Lys biosynthetic pathway in T. maritima. The Lys racemase had a dimeric structure containing pyridoxal 5'-phosphate as a cofactor. Among the amino acids, it exhibited the highest racemase activity toward d- and l-Lys, and also had relatively high activity toward d- and l-enantiomers of ornithine and Ala. The Dpm epimerase had the highest epimerization activity toward ll- and meso-Dpm, and also measurably racemized certain amino acids, including Lys. These results suggest that Lys racemase contributes to production of d-Lys and d-Ala for use as peptidoglycan components, and that Dpm epimerase converts ll-Dpm to meso-Dpm, a precursor in the l-Lys biosynthetic pathway.


Assuntos
Isomerases de Aminoácido/metabolismo , Parede Celular/enzimologia , Regulação Bacteriana da Expressão Gênica , Lisina/biossíntese , Thermotoga maritima/enzimologia , Alanina/química , Alanina/metabolismo , Isomerases de Aminoácido/genética , Sequência de Aminoácidos , Parede Celular/química , Clonagem Molecular , Coenzimas/química , Coenzimas/metabolismo , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Redes e Vias Metabólicas , Ornitina/química , Ornitina/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Multimerização Proteica , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato , Thermotoga maritima/química , Thermotoga maritima/genética
11.
Biomacromolecules ; 20(2): 846-853, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30521331

RESUMO

In vitro polymerization of ß-mannans is a challenging reaction due to the steric hindrance confered by the configuration of mannosyl residues and the thermodynamic instability of the ß-anomer. Whatever the approach used to date-whether chemical, or enzymatic with glycosynthases and mannosyltransferases-pure ß-1,4-mannans have never been synthesized in vitro. This has limited attempts to investigate their role in the production of plant and algal cell walls, in which they are highly abundant. It has also impeded the exploitation of their properties as biosourced materials. In this paper, we demonstrate that TM1225, a thermoactive glycoside phosphorylase from the hyperthermophile species Thermotoga maritima, is a powerful biocatalytic tool for the ecofriendly synthesis of pure ß-1,4-mannan. The recombinant production of this enzyme and its biochemical characterization allowed us to prove that it catalyzes the reversible phosphorolysis of ß-1,4-mannosides, and determine its role in the metabolism of the algal mannans on which T. maritima feeds in submarine sediments. Furthermore, after optimizing the reaction conditions, we exploited the synthetic ability of TM1225 to produce ß-1,4-mannan in vitro. At 60 °C and from d-mannose 1-phosphate and mannohexaose, the enzyme synthesized mannoside chains with a degree of polymerization up to 16, which precipitated into lamellar single crystals. The X-ray powder diffraction and base-plane electron diffraction patterns of the lamellar crystals unambiguously show that the synthesized product belongs to the mannan I family previously observed in planta in pure linear mannans, such as those of the ivory nut. The in vitro formation of these mannan I crystals is likely determined by the high reaction temperature and the narrow chain length distribution of the insoluble chains.


Assuntos
Biocatálise , Mananas/síntese química , Proteínas de Bactérias/metabolismo , Cristalização , Fosforilases/metabolismo , Polimerização , Thermotoga maritima/enzimologia
12.
Protein Sci ; 28(1): 267-282, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30394621

RESUMO

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA• and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Sulfurtransferases/metabolismo , Thermotoga maritima/enzimologia , Transporte de Elétrons/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Sulfurtransferases/genética , Thermotoga maritima/genética
13.
Nat Commun ; 9(1): 5381, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568191

RESUMO

In order to preserve genomic stability, cells rely on various repair pathways for removing DNA damage. The mechanisms how enzymes scan DNA and recognize their target sites are incompletely understood. Here, by using high-localization precision microscopy along with 133 Hz high sampling rate, we have recorded EndoV and OGG1 interacting with 12-kbp elongated λ-DNA in an optical trap. EndoV switches between three distinct scanning modes, each with a clear range of activation energy barriers. These results concur with average diffusion rate and occupancy of states determined by a hidden Markov model, allowing us to infer that EndoV confinement occurs when the intercalating wedge motif is involved in rigorous probing of the DNA, while highly mobile EndoV may disengage from a strictly 1D helical diffusion mode and hop along the DNA. This makes EndoV the first example of a monomeric, single-conformation and single-binding-site protein demonstrating the ability to switch between three scanning modes.


Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Thermotoga maritima/enzimologia , DNA Glicosilases/metabolismo , Escherichia coli , Cadeias de Markov , Imagem Individual de Molécula , Thermotoga maritima/genética
14.
J Phys Chem B ; 122(46): 10490-10495, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30365331

RESUMO

Enzymes exist in continuously fluctuating water bath dramatically affecting their function. Water not only forms the solvation shell but also penetrates into the protein interior. Changing the wetting pattern of the protein's active site in response to altering redox state initiates a highly nonlinear structural change and non-Gaussian electrostatic fluctuations at the active site. The free-energy surfaces of electron transfer are highly nonparabolic (non-Marcusian), as shown by atomistic molecular dynamics simulations of hydrated ferredoxin protein and by an analytical model in agreement with simulations. The reorganization energy of electron transfer passes through a spike marking equal probabilities of the wet and dry states of the active site. The activation thermodynamics affected by wetting leads to a non-Arrhenius, passing through a maximum, plot for the reaction rate vs the inverse temperature.


Assuntos
Proteínas de Bactérias/metabolismo , Ferredoxinas/metabolismo , Água/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , Elétrons , Ferredoxinas/química , Cinética , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , Temperatura , Termodinâmica , Thermotoga maritima/enzimologia , Água/química
15.
Enzyme Microb Technol ; 119: 1-9, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30243380

RESUMO

Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia coli (Aeromonas jandaeil-allo-threonine aldolase, E. colil-threonine aldolase and Thermotoga maritimal-allo-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme. These conditions were used to characterize the substrate- and stereoselectivity of each TA toward a panel of aldehyde acceptors. In general, the A. jandaei TA performed best, and six representative conversions were scaled up 10-fold in order to develop downstream steps for product isolation. One key improvement was to treat the crude reaction product with Bacillus subtilis glycine oxidase, which eliminated residual starting material and significantly simplified product isolation. NMR studies were used to identify the major and minor diastereomers from the preparative-scale reactions and the absolute configurations for three representative cases.


Assuntos
Aeromonas/enzimologia , Escherichia coli/enzimologia , Glicina Hidroximetiltransferase/metabolismo , Thermotoga maritima/enzimologia , Aldeídos/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Bacillus subtilis/enzimologia , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/isolamento & purificação , Especificidade por Substrato , Treonina/metabolismo
16.
Biosci Biotechnol Biochem ; 82(12): 2084-2093, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30175674

RESUMO

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 µM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 µM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK-HseDH: bifunctional aspartate kinase-homoserine dehydrogenase; AsaDH: aspartate-ß-semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).


Assuntos
Aspartoquinase Homosserina Desidrogenase/metabolismo , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Aspartoquinase Homosserina Desidrogenase/química , Aspartoquinase Homosserina Desidrogenase/genética , Biocatálise , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Treonina/metabolismo
17.
Biomolecules ; 8(3)2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-30061529

RESUMO

Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2⁻X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites -2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.


Assuntos
Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Simulação de Acoplamento Molecular , Temperatura , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Estabilidade Enzimática , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Ligação Proteica , Especificidade por Substrato
18.
Extremophiles ; 22(6): 889-894, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30088105

RESUMO

The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-L-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-L-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.


Assuntos
Proteínas de Bactérias/metabolismo , Oligossacarídeos/biossíntese , Thermotoga maritima/enzimologia , alfa-L-Fucosidase/metabolismo , Cálcio/metabolismo , Fucose/análogos & derivados , Sódio/metabolismo
19.
Nat Commun ; 9(1): 3243, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104598

RESUMO

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate's structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.


Assuntos
Carbaçúcares/farmacologia , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Biocatálise , Carbaçúcares/síntese química , Carbaçúcares/química , Domínio Catalítico , Cicloexenos/síntese química , Cicloexenos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Galactose/análogos & derivados , Glicosídeo Hidrolases/química , Cinética , Simulação de Dinâmica Molecular , Teoria Quântica , Thermotoga maritima/enzimologia
20.
J Mol Biol ; 430(22): 4592-4602, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30044948

RESUMO

AAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (TmFtsH). Using a single-molecule Förster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that TmFtsH-even in the absence of nucleotide-is a highly dynamic protease undergoing sequential transitions between five states on the second timescale. Addition of ATP does not influence the number of states or change the timescale of domain motions but affects the state occupancy distribution leading to an inter-domain compaction. These findings suggest that thermal energy, but not chemical energy, provides the major driving force for conformational switching, while ATP, through a state reequilibration, introduces directionality into this process. The TmFtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin (SPG7) causing hereditary spastic paraplegia, does not affect the dynamic behavior of the protease but impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in hereditary spastic paraplegia.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Thermotoga maritima/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Bactérias/química , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Imagem Individual de Molécula , Termodinâmica , Thermotoga maritima/genética
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