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1.
Phys Chem Chem Phys ; 23(23): 13042-13054, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34100037

RESUMO

Many enzyme reactions present instantaneous disorder. These dynamic fluctuations in the enzyme-substrate Michaelis complexes generate a wide range of energy barriers that cannot be experimentally observed, but that determine the measured kinetics of the reaction. These individual energy barriers can be calculated using QM/MM methods, but then the problem is how to deal with this dispersion of energy barriers to provide kinetic information. So far, the most usual procedure has implied the so-called exponential average of the energy barriers. In this paper, we discuss the foundations of this method, and we use the free energy perturbation theory to derive an alternative equation to get the Gibbs free energy barrier of the enzyme reaction. In addition, we propose a practical way to implement it. We have chosen four enzyme reactions as examples. In particular, we have studied the hydrolysis of a glycosidic bond catalyzed by the enzyme Thermus thermophilus ß-glycosidase, and the mutant Y284P Ttb-gly, and the hydrogen abstraction reactions from C13 and C7 of arachidonic acid catalyzed by the enzyme rabbit 15-lipoxygenase-1.


Assuntos
Araquidonato 15-Lipoxigenase/química , Teoria da Densidade Funcional , Glicosídeo Hidrolases/química , Termodinâmica , Thermus thermophilus/enzimologia , Animais , Araquidonato 15-Lipoxigenase/metabolismo , Glicosídeo Hidrolases/metabolismo , Cinética , Coelhos
2.
Nat Chem Biol ; 17(7): 800-805, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958791

RESUMO

The covalent attachment of one or multiple heme cofactors to cytochrome c protein chains enables cytochrome c proteins to be used in electron transfer and redox catalysis in extracytoplasmic environments. A dedicated heme maturation machinery, whose core component is a heme lyase, scans nascent peptides after Sec-dependent translocation for CXnCH-binding motifs. Here we report the three-dimensional (3D) structure of the heme lyase CcmF, a 643-amino acid integral membrane protein, from Thermus thermophilus. CcmF contains a heme b cofactor at the bottom of a large cavity that opens toward the extracellular side to receive heme groups from the heme chaperone CcmE for cytochrome maturation. A surface groove on CcmF may guide the extended apoprotein to heme attachment at or near a loop containing the functionally essential WXWD motif, which is situated above the putative cofactor binding pocket. The structure suggests heme delivery from within the membrane, redefining the role of the chaperone CcmE.


Assuntos
Membrana Celular/metabolismo , Liases/metabolismo , Membrana Celular/química , Liases/química , Thermus thermophilus/enzimologia
3.
Appl Environ Microbiol ; 87(14): e0054121, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33990309

RESUMO

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for the high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using CoA-insensitive type III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using design of experiments (DOE) was employed to rationally determine the enzyme loading ratio to maximize the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK toward ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type I and type II pantothenate kinase (PanK). Type III PanK is found only in bacteria and is generally insensitive to CoA. Previously, type III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic and instead shows inhibition toward CoA. In the present study, phylogenetic analysis combined with functional analysis of type III PanK from thermophiles revealed that the CoA-sensitive behavior of type III PanK from T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and Geobacillus sp. strain 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade reaction to produce CoA.


Assuntos
Proteínas de Bactérias/metabolismo , Coenzima A/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Thermus thermophilus/enzimologia
4.
Photochem Photobiol Sci ; 20(5): 663-670, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33977512

RESUMO

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.


Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas de Ligação ao GTP , Processos Fotoquímicos
5.
Angew Chem Int Ed Engl ; 60(32): 17680-17685, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34056805

RESUMO

ß-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic ß-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3 -rich cyclic fragments and the first total synthesis of jomthonic acid A.


Assuntos
Aminoácidos Aromáticos/síntese química , Aminoácidos de Cadeia Ramificada/síntese química , Aminação , Aminoácidos/síntese química , Proteínas Arqueais/química , Proteínas de Bactérias/química , Biocatálise , Pyrococcus horikoshii/enzimologia , Estereoisomerismo , Thermococcus/enzimologia , Thermus thermophilus/enzimologia , Transaminases/química
6.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 5): 148-155, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33949975

RESUMO

Fatty-acid degradation is an oxidative process that involves four enzymatic steps and is referred to as the ß-oxidation pathway. During this process, long-chain acyl-CoAs are broken down into acetyl-CoA, which enters the mitochondrial tricarboxylic acid (TCA) cycle, resulting in the production of energy in the form of ATP. Enoyl-CoA hydratase (ECH) catalyzes the second step of the ß-oxidation pathway by the syn addition of water to the double bond between C2 and C3 of a 2-trans-enoyl-CoA, resulting in the formation of a 3-hydroxyacyl CoA. Here, the crystal structure of ECH from Thermus thermophilus HB8 (TtECH) is reported at 2.85 Šresolution. TtECH forms a hexamer as a dimer of trimers, and wide clefts are uniquely formed between the two trimers. Although the overall structure of TtECH is similar to that of a hexameric ECH from Rattus norvegicus (RnECH), there is a significant shift in the positions of the helices and loops around the active-site region, which includes the replacement of a longer α3 helix with a shorter α-helix and 310-helix in RnECH. Additionally, one of the catalytic residues of RnECH, Glu144 (numbering based on the RnECH enzyme), is replaced by a glycine in TtECH, while the other catalytic residue Glu164, as well as Ala98 and Gly141 that stabilize the enolate intermediate, is conserved. Their putative ligand-binding sites and active-site residue compositions are dissimilar.


Assuntos
Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/metabolismo , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Homologia de Sequência
7.
Nucleic Acids Res ; 49(9): 5351-5368, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33885823

RESUMO

Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.


Assuntos
Mycobacterium tuberculosis/enzimologia , Fenilalanina-tRNA Ligase/química , RNA de Transferência de Fenilalanina/química , Aminoacilação de RNA de Transferência , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Mycobacterium tuberculosis/genética , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina-tRNA Ligase/metabolismo , Ligação Proteica , RNA de Transferência de Fenilalanina/metabolismo , Thermus thermophilus/enzimologia
8.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799806

RESUMO

Carbonic anhydrases (CAs) have been identified as ideal catalysts for CO2 sequestration. Here, we report the sequence and structural analyses as well as the molecular dynamics (MD) simulations of four γ-CAs from thermophilic bacteria. Three of these, Persephonella marina, Persephonella hydrogeniphila, and Thermosulfidibacter takaii originate from hydrothermal vents and one, Thermus thermophilus HB8, from hot springs. Protein sequences were retrieved and aligned with previously characterized γ-CAs, revealing differences in the catalytic pocket residues. Further analysis of the structures following homology modeling revealed a hydrophobic patch in the catalytic pocket, presumed important for CO2 binding. Monitoring of proton shuttling residue His69 (P. marina γ-CA numbering) during MD simulations of P. hydrogeniphila and P. marina's γ-CAs (γ-PhCA and γ-PmCA), showed a different behavior to that observed in the γ-CA of Escherichia coli, which periodically coordinates Zn2+. This work also involved the search for hotspot residues that contribute to interface stability. Some of these residues were further identified as key in protein communication via betweenness centrality metric of dynamic residue network analysis. T. takaii's γ-CA showed marginally lower thermostability compared to the other three γ-CA proteins with an increase in conformations visited at high temperatures being observed. Hydrogen bond analysis revealed important interactions, some unique and others common in all γ-CAs, which contribute to interface formation and thermostability. The seemingly thermostable γ-CA from T. thermophilus strangely showed increased unsynchronized residue motions at 423 K. γ-PhCA and γ-PmCA were, however, preliminarily considered suitable as prospective thermostable CO2 sequestration agents.


Assuntos
Proteínas de Bactérias/metabolismo , Biomineralização , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dióxido de Carbono/química , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Catálise , Domínio Catalítico , Simulação por Computador , Fontes Termais/microbiologia , Fontes Hidrotermais/microbiologia , Simulação de Dinâmica Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Thermus thermophilus/enzimologia
9.
Biomolecules ; 11(4)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923608

RESUMO

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.


Assuntos
Proteínas de Bactérias/metabolismo , Nucleosídeos/biossíntese , Pentosiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases/metabolismo , Thermus thermophilus/metabolismo , Escherichia coli/metabolismo , Pentoses/metabolismo , Thermus thermophilus/enzimologia
10.
Biochim Biophys Acta Gen Subj ; 1865(7): 129895, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33781823

RESUMO

BACKGROUND: Understanding the determinants of protein thermostability is very important both from the theoretical and applied perspective. One emerging view in thermostable enzymes seems to indicate that a salt bridge/charged residue network plays a fundamental role in their thermostability. METHODS: The structure of alkaline phosphatase (AP) from Thermus thermophilus HB8 was solved by X-ray crystallography at 2.1 Å resolution. The obtained structure was further analyzed by molecular dynamics studies at different temperatures (303 K, 333 K and 363 K) and compared to homologous proteins from the cold-adapted organisms Shewanella sp. and Vibrio strain G15-21. To analyze differences in measures of dynamic variation, several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis were used. Using hierarchical clustering, the obtained results were combined to determine residues showing high degree dynamical variations due to temperature jumps. Furthermore, dynamic cross correlation (DCC) analysis was carried out to characterize networks of charged residues. RESULTS: Top clustered residues showed a higher propensity for thermostabilizing mutations, indicating evolutionary pressure acting on thermophilic organisms. The description of rotamer distributions by Gini coefficients and Kullback-Leibler (KL) divergence both revealed significant correlations with temperature. DCC analysis revealed a significant trend to de-correlation of the movement of charged residues at higher temperatures. SIGNIFICANCE: The de-correlation of charged residues detected in Thermus thermophilus AP, highlights the importance of dynamic electrostatic network interactions for the thermostability of this enzyme.


Assuntos
Fosfatase Alcalina/química , Temperatura Alta , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Estabilidade Enzimática , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação Proteica , Homologia de Sequência
11.
Biochimie ; 182: 228-237, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33535124

RESUMO

Thermus thermophilus laccase belongs to the sub-class of multicopper oxidases that is activated by the extra binding of copper to a methionine-rich domain allowing an electron pathway from the substrate to the conventional first electron acceptor, the T1 Cu. In this work, two key amino acid residues in the 1st and 2nd coordination spheres of T1 Cu are mutated in view of tuning their redox potential and investigating their influence on copper-related activity. Evolution of the kinetic parameters after copper addition highlights that both mutations play a key role influencing the enzymatic activity in distinct unexpected ways. These results clearly indicate that the methionine rich domain is not the only actor in the cuprous oxidase activity of CueO-like enzymes.


Assuntos
Proteínas de Bactérias/química , Cobre/química , Lacase/química , Mutação , Thermus thermophilus/enzimologia , Proteínas de Bactérias/genética , Lacase/genética
12.
Biochim Biophys Acta Gen Subj ; 1865(6): 129872, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33588000

RESUMO

BACKGROUND: Laccase is one member of the blue multicopper oxidase family. It can catalyze the oxidation of various substrates. The Thermus thermophilus SG0.5JP17-16 laccase (lacTT) is thermostable, pH-stable, and high tolerance to halides, and can decolorize the synthetic dyes. In lacTT, the function of the loop 6 constructing the substrate-binding pocket wasn't clear. METHODS: The residues Asp394 and Asp396 located in loop 6, and were used to probe how the loop 6 influenced catalytic properties of the laccase. Site-directed mutagenesis was performed for two amino acids. Kinetic assay was utilized to characterize the catalytic efficiency of mutants. Mutants with different catalytic activities were used to decolorize the synthetic dyes to clarify the relationship between the catalytic efficiency and dye decolorization. Redox potential, structural and spectral analyses were performed to explain the differences in laccase activity between wild type and mutant enzymes. RESULTS: D394M, D394E and D394R mutants with the lower laccase activity displayed a decreased decolorization efficiency, while D396A, D396M and D396E mutant enzymes with higher catalytic efficiency decolorized the synthetic dye more efficiently than the wild type enzyme. CONCLUSIONS: The pocket loop 6 might experience a conformational dynamics. The D394 residue controlled this conformation change by amino acid interaction networks containing the D396 residue at the entrance of substrate channel. GENERAL SIGNIFICANCES: These studies may provide clues to improve the activity of the laccase for the better use in industrial applications, and/or contribute to further understanding the mechanism of laccase oxidation on the substrate.


Assuntos
Proteínas de Bactérias/metabolismo , Lacase/metabolismo , Mutação , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Lacase/química , Lacase/genética , Mutagênese Sítio-Dirigida , Conformação Proteica
13.
Nat Commun ; 12(1): 796, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542236

RESUMO

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
14.
Sci Rep ; 11(1): 2991, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542380

RESUMO

The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol-1 for ArsC-Vmh2 and to 1200 (± 300) L mol-1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).


Assuntos
Arseniato Redutases/química , Arsênio/isolamento & purificação , Técnicas Biossensoriais , Proteínas Fúngicas/química , Proteínas Recombinantes de Fusão/química , Arsênio/toxicidade , Enzimas Imobilizadas/química , Escherichia coli/genética , Humanos , Pleurotus/química , Pleurotus/enzimologia , Thermus thermophilus/enzimologia
15.
Microbiologyopen ; 10(1): e1149, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33415847

RESUMO

Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.


Assuntos
DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Thermus/enzimologia , Thermus/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Polimerase I/química , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Taq Polimerase/genética , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
16.
FEBS Lett ; 595(2): 264-274, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33159808

RESUMO

Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand-induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP-binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins.


Assuntos
Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Thermus thermophilus/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Estabilidade Enzimática , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/genética , Proteólise , Thermus thermophilus/genética , Temperatura de Transição
17.
Biochim Biophys Acta Bioenerg ; 1862(1): 148326, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33045211

RESUMO

The structure of the entire respiratory complex I is now known at reasonably high resolution for many species - bacteria, yeast, and several mammals, including human. The structure reveals an almost 30 angstrom tunnel-like chamber for ubiquinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. Here we characterize the geometric bottleneck forming the entrance of the quinone reaction chamber. Computer simulations of quinone/quinol passage through the bottleneck suggest that in all structures available, from bacterial to human, this bottleneck is too narrow for the quinone or quinol to pass and that a conformational change is required to open the channel. Moreover, the bottleneck is too narrow even for isoprenoid tail free passage. The closed structure can be an artifact of the crystallization packing forces, low temperature, or other unnatural conditions occurring in the structural data acquisition procedure that affect this flexible part of the enzyme. Two of the helices forming the bottleneck are in direct contact with the subunit (ND3) that was recently demonstrated to be involved in conformational changes during the redox proton pumping cycle, which indicates flexibility of that part of the enzyme. We conclude that the published structures are all locked in the unfunctional states and do not represent correctly the functional enzyme; we discuss possible ways to open the structure in the context of possible mechanisms of the enzyme.


Assuntos
Proteínas de Bactérias/química , Benzoquinonas/química , Complexo I de Transporte de Elétrons/química , Proteínas Fúngicas/química , Thermus thermophilus/enzimologia , Yarrowia/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Ovinos
18.
ACS Appl Mater Interfaces ; 12(50): 56027-56038, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33275418

RESUMO

Understanding how the immobilization of enzymes on solid carriers affects their performance is paramount for the design of highly efficient heterogeneous biocatalysts. An efficient supply of substrates onto the solid phase is one of the main challenges to maximize the activity of the immobilized enzymes. Herein, we apply advanced single-particle analysis to decipher the optimal design of an immobilized NADH oxidase (NOX) whose activity depends both on O2 and NADH concentrations. Carrier physicochemical properties and its functionality along with the enzyme distribution across the carrier were implemented as design variables to study the effects of the intraparticle concentration of substrates (O2 and NADH) on the activity. Intraparticle O2-sensing analysis revealed the superior performance of the enzyme immobilized at the outer surface in terms of effective supply of O2. Furthermore, the co-immobilization of NADH and NOX within the tuned surface of porous microbeads increases the effective concentration of NADH in the surroundings of the enzyme. As a result, the optimal spatial organization of NOX and its confinement with NADH allow a 100% recovery of the activity of the soluble enzyme upon the immobilization process. By engineering these variables, we increase the NADH oxidation activity of the heterogeneous biocatalyst by up to 650% compared to NOX immobilized under suboptimal conditions. In conclusion, this work highlights the rational design and engineering of the enzyme-carrier interface to maximize the efficiency of heterogeneous biocatalysts.


Assuntos
Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , NAD/metabolismo , Oxigênio/metabolismo , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Complexos Multienzimáticos/química , NADH NADPH Oxirredutases/química , Sefarose/química , Especificidade por Substrato , Thermus thermophilus/enzimologia
19.
Sci Rep ; 10(1): 21024, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273609

RESUMO

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Microbiologia Industrial/métodos , Thermus thermophilus/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo
20.
J Chem Phys ; 153(13): 134104, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33032427

RESUMO

Molecular interactions are essential for regulation of cellular processes from the formation of multi-protein complexes to the allosteric activation of enzymes. Identifying the essential residues and molecular features that regulate such interactions is paramount for understanding the biochemical process in question, allowing for suppression of a reaction through drug interventions or optimization of a chemical process using bioengineered molecules. In order to identify important residues and information pathways within molecular complexes, the dynamical network analysis method was developed and has since been broadly applied in the literature. However, in the dawn of exascale computing, this method is frequently limited to relatively small biomolecular systems. In this work, we provide an evolution of the method, application, and interface. All data processing and analysis are conducted through Jupyter notebooks, providing automatic detection of important solvent and ion residues, an optimized and parallel generalized correlation implementation that is linear with respect to the number of nodes in the system, and subsequent community clustering, calculation of betweenness of contacts, and determination of optimal paths. Using the popular visualization program visual molecular dynamics (VMD), high-quality renderings of the networks over the biomolecular structures can be produced. Our new implementation was employed to investigate three different systems, with up to 2.5M atoms, namely, the OMP-decarboxylase, the leucyl-tRNA synthetase complexed with its cognate tRNA and adenylate, and respiratory complex I in a membrane environment. Our enhanced and updated protocol provides the community with an intuitive and interactive interface, which can be easily applied to large macromolecular complexes.


Assuntos
Complexo I de Transporte de Elétrons/química , Leucina-tRNA Ligase/química , Orotidina-5'-Fosfato Descarboxilase/química , Regulação Alostérica , Domínio Catalítico , Escherichia coli/enzimologia , Methanobacteriaceae/enzimologia , Simulação de Dinâmica Molecular , Domínios Proteicos , Software , Thermus thermophilus/enzimologia
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