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1.
Nat Commun ; 12(1): 4849, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381032

RESUMO

Although various artificial protein nanoarchitectures have been constructed, controlling the transformation between different protein assemblies has largely been unexplored. Here, we describe an approach to realize the self-assembly transformation of dimeric building blocks by adjusting their geometric arrangement. Thermotoga maritima ferritin (TmFtn) naturally occurs as a dimer; twelve of these dimers interact with each other in a head-to-side manner to generate 24-meric hollow protein nanocage in the presence of Ca2+ or PEG. By tuning two contiguous dimeric proteins to interact in a fully or partially side-by-side fashion through protein interface redesign, we can render the self-assembly transformation of such dimeric building blocks from the protein nanocage to filament, nanorod and nanoribbon in response to multiple external stimuli. We show similar dimeric protein building blocks can generate three kinds of protein materials in a manner that highly resembles natural pentamer building blocks from viral capsids that form different protein assemblies.


Assuntos
Nanoestruturas/química , Proteínas/química , Cálcio/química , Ferritinas/química , Nanoestruturas/ultraestrutura , Nanotecnologia , Polietilenoglicóis/química , Multimerização Proteica , Thermotoga maritima
2.
Nat Commun ; 12(1): 4542, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315871

RESUMO

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.


Assuntos
Formaldeído/metabolismo , Nucleotídeos/metabolismo , Thermotoga maritima/enzimologia , Timidilato Sintase/metabolismo , Biocatálise , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Domínio Catalítico , Ativação Enzimática , Flavinas/metabolismo , Metilação , Eletricidade Estática , Timidilato Sintase/química
3.
Nanoscale ; 13(27): 11932-11942, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34195748

RESUMO

Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium Thermotoga maritima (TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like Archeoglobus fulgidus ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg2+). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.


Assuntos
Ferritinas , Ferro , Ferritinas/genética , Ferro/metabolismo , Thermotoga maritima
4.
J Ind Microbiol Biotechnol ; 48(5-6)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34124750

RESUMO

Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a ß-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-ß-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent kcat, respectively; N222F shows the highest kcat/km value for pNPG. The substrate selectivity altered from pNPG to pNP-ß-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-ß-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis.


Assuntos
Glicosídeos/biossíntese , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Alquilação , Dissacarídeos/biossíntese , Glicosídeo Hidrolases/metabolismo , Hidrólise , Microbiologia Industrial , Cinética , Engenharia Metabólica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Especificidade por Substrato
5.
Appl Environ Microbiol ; 87(14): e0052421, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33990300

RESUMO

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCE Caldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.


Assuntos
Actinobacteria/enzimologia , Caldicellulosiruptor/metabolismo , Proteoma/metabolismo , Thermotoga maritima/enzimologia , Xilanos/metabolismo , Xilosidases/metabolismo , Actinobacteria/genética , Celobiose/metabolismo , Escherichia coli/genética , Thermotoga maritima/genética , Xilosidases/genética
6.
J Biol Chem ; 296: 100797, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019879

RESUMO

Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree. In this pathway, most bacteria use an O-acetylhomoserine aminocarboxypropyltransferase (MetY) to catalyze the formation of homocysteine from O-acetylhomoserine and bisulfide. Despite the widespread distribution of MetY, this pyridoxal 5'-phosphate-dependent enzyme remains comparatively understudied. To address this knowledge gap, we have characterized the MetY from Thermotoga maritima (TmMetY). At its optimal temperature of 70 °C, TmMetY has a turnover number (apparent kcat = 900 s-1) that is 10- to 700-fold higher than the three other MetY enzymes for which data are available. We also present crystal structures of TmMetY in the internal aldimine form and, fortuitously, with a ß,γ-unsaturated ketimine reaction intermediate. This intermediate is identical to that found in the catalytic cycle of cystathionine γ-synthase (MetB), which is a homologous enzyme from the trans-sulfurylation pathway. By comparing the TmMetY and MetB structures, we have identified Arg270 as a critical determinant of specificity. It helps to wall off the active site of TmMetY, disfavoring the binding of the first MetB substrate, O-succinylhomoserine. It also ensures a strict specificity for bisulfide as the second substrate of MetY by occluding the larger MetB substrate, cysteine. Overall, this work illuminates the subtle structural mechanisms by which homologous pyridoxal 5'-phosphate-dependent enzymes can effect different catalytic, and therefore metabolic, outcomes.


Assuntos
Proteínas de Bactérias/metabolismo , Metionina/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Bactérias/química , Vias Biossintéticas , Cristalografia por Raios X , Cinética , Modelos Moleculares , Thermotoga maritima/química
7.
Extremophiles ; 25(3): 311-317, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33938983

RESUMO

Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants' health. These compounds can be also obtained by enzymatic synthesis. In this work, the effect of the immobilization of α-L-fucosidase from Thermotoga maritima on the synthesis of fucosylated oligosaccharides was studied, using lactose and 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as acceptor and donor substrates, respectively, and Eupergit® CM as an immobilization support. The enzyme was immobilized with 90% efficiency at pH 8 and ionic strength of 1.5 M. Immobilization decreased enzyme affinity for the donor substrate as shown by a 1.5-times higher KM value and a 22-times decrease of the kcat/KM ratio in comparison to the unbound enzyme. In contrast, no effect was observed on the synthesis/hydrolysis ratio (rs/rh) when α-L-fucosidase was immobilized. Also, the effect of initial concentration of substrates was studied. An increase of the acceptor concentration improved the yields of fucosylated oligosaccharides regardless enzyme immobilization. The synthesis yields of 38.9 and 40.6% were obtained using Eupergit® CM-bound or unbound enzyme, respectively, and 3.5 mM pNP-Fuc and 146 mM lactose. In conclusion, α-L-fucosidase from Thermotoga maritima was efficiently immobilized on Eupergit® CM support without affecting the synthesis of fucosylated oligosaccharides.


Assuntos
Thermotoga maritima , alfa-L-Fucosidase , Fucose , Oligossacarídeos , Especificidade por Substrato , Thermotoga , Thermotoga maritima/metabolismo , alfa-L-Fucosidase/metabolismo
8.
Nat Commun ; 12(1): 2748, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980881

RESUMO

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.


Assuntos
Regulação Alostérica , Aminoidrolases/química , Aminoidrolases/metabolismo , Aminoidrolases/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glutamina/metabolismo , Hidrólise , Imidazóis/metabolismo , Espectroscopia de Ressonância Magnética , Complexos Multienzimáticos , Mutação , Conformação Proteica , Ribonucleotídeos/metabolismo , Thermotoga maritima/enzimologia
9.
Biochemistry (Mosc) ; 86(4): 397-408, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33941062

RESUMO

Ribosomal protein L1 is a conserved two-domain protein that is involved in formation of the L1 stalk of the large ribosomal subunit. When there are no free binding sites available on the ribosomal 23S RNA, the protein binds to the specific site on the mRNA of its own operon (L11 operon in bacteria and L1 operon in archaea) preventing translation. Here we show that the regulatory properties of the r-protein L1 and its domain I are conserved in the thermophilic bacteria Thermus and Thermotoga and in the halophilic archaeon Haloarcula marismortui. At the same time the revealed features of the operon regulation in thermophilic bacteria suggest presence of two regulatory regions.


Assuntos
Haloarcula marismortui/genética , Óperon/genética , Sequências Reguladoras de Ácido Nucleico , Proteínas Ribossômicas/genética , Thermotoga maritima/genética , Thermus thermophilus/genética , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Haloarcula marismortui/metabolismo , Temperatura Alta , Thermotoga maritima/metabolismo , Thermus thermophilus/metabolismo
10.
ACS Chem Biol ; 16(5): 794-799, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33877806

RESUMO

Most kinases utilize ATP as a phosphate donor and phosphorylate a wide range of phosphate acceptors. An alternative phosphate donor is inorganic pyrophosphate (PPi), which costs only 1/1000 of ATP. To develop a method to engineer PPi-dependent kinases, we herein aimed to alter the product of PPi-dependent myo-inositol kinase from d-myo-inositol 1-phosphate to d-myo-inositol 3-phosphate. For this purpose, we introduced the myo-inositol recognition residues of the ATP-dependent myo-inositol-3-kinase into the PPi-dependent myo-inositol-1-kinase. This replacement was expected to change the 3D arrangements of myo-inositol in the active site and bring the hydroxyl group at the 3C position close to the catalytic residue. LC-MS and NMR analyses proved that the engineered enzyme successfully produced myo-inositol 3-phosphate from PPi and myo-inositol.


Assuntos
Difosfatos/química , Monoéster Fosfórico Hidrolases/química , Thermotoga maritima/enzimologia , Domínio Catalítico , Cristalização , Fosfatos de Inositol/química , Cinética , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Mutação , Fosforilação , Conformação Proteica , Espectrometria de Massas em Tandem
11.
Biochem J ; 478(4): 943-959, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33565573

RESUMO

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and ß-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Šresolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.


Assuntos
Proteínas de Bactérias/química , Apoenzimas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ditiotreitol/metabolismo , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Glicosídeo Hidrolases/metabolismo , Holoenzimas/química , Cinética , Manganês/metabolismo , Modelos Moleculares , Família Multigênica , Mutagênese Sítio-Dirigida , NAD/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Thermotoga maritima/enzimologia , Thermotoga maritima/genética
12.
Biochim Biophys Acta Proteins Proteom ; 1869(4): 140602, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33422670

RESUMO

Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with 1H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in 1H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.


Assuntos
Proteínas de Bactérias/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Bactérias/química , Catálise , Domínio Catalítico , Glucose-6-Fosfato Isomerase/química , Isomerismo , Cinética , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
13.
J Am Chem Soc ; 143(1): 335-348, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33372786

RESUMO

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , S-Adenosilmetionina/química , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Biocatálise , Domínio Catalítico , Clostridium acetobutylicum/enzimologia , Teoria da Densidade Funcional , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/efeitos da radiação , Luz , Modelos Químicos , Estrutura Molecular , Oxirredução/efeitos da radiação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/efeitos da radiação , Fotólise , S-Adenosilmetionina/efeitos da radiação , Thermotoga maritima/enzimologia
14.
Methods Mol Biol ; 2253: 137-151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315222

RESUMO

Community network analysis (CNA) of correlated protein motions allows modeling of signals propagation in allosteric proteic systems. From standard classical molecular dynamics (MD) simulations, protein motions can be analysed by means of mutual information between pairs of amino acid residues, providing dynamical weighted networks that contains fundamental information of the communication among amino acids. The CNA method has been successfully applied to a variety of allosteric systems including an enzyme, a nuclear receptor and a bacterial adaptive immune system, providing characterization of the allosteric pathways. This method is complementary to network analyses based on different metrics and it is particularly powerful for studying large proteic systems, as it provides a coarse-grained view of the communication flows within large and complex networks.


Assuntos
Aminoidrolases/química , Aminoidrolases/metabolismo , Thermotoga maritima/enzimologia , Regulação Alostérica , Sítio Alostérico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Mapas de Interação de Proteínas , Thermotoga maritima/química
15.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140523, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853774

RESUMO

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Thermotoga maritima/química , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Thermotoga maritima/enzimologia
16.
Nat Commun ; 11(1): 5644, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159067

RESUMO

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase ß-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Thermotoga maritima/enzimologia , Triptofano Sintase/química , Proteínas de Bactérias/genética , Biocatálise , Evolução Molecular , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Thermotoga maritima/química , Thermotoga maritima/genética , Triptofano/química , Triptofano/metabolismo , Triptofano Sintase/genética , Triptofano Sintase/metabolismo
17.
PLoS One ; 15(10): e0241557, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33126240

RESUMO

Metallo-ß-lactamases (MBLs) hydrolyze a wide range of ß-lactam antibiotics. While all MBLs share a common αß/ßα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily. Thermotoga maritima tRNase Z, a tRNA 3' processing endoribonuclease of MBL-superfamily, and IMP-1, a clinically isolated MBL, showed a striking similarity in tertiary structure, despite low sequence homology. IMP-1 hydrolyzed both total cellular RNA and synthetic small unstructured RNAs. IMP-1 also hydrolyzed pre-tRNA, but its cleavage site was different from those of T. maritima tRNase Z and human tRNase Z long form, indicating a key difference in substrate recognition. Single-turnover kinetic assays suggested that substrate-binding affinity of T. maritima tRNase Z is much higher than that of IMP-1.


Assuntos
RNA/metabolismo , Thermotoga maritima/enzimologia , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Humanos , Hidrólise , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Thermotoga maritima/química , Thermotoga maritima/metabolismo , beta-Lactamases/química
18.
Protein J ; 39(5): 487-500, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33094361

RESUMO

Prokaryotic cold shock proteins (CSPs) are considered to play an important role in the transcriptional and translational regulation of gene expression, possibly by acting as transcription anti-terminators and "RNA chaperones". They bind with high affinity to single-stranded nucleic acids. Here we report the binding epitope of TmCsp from Thermotoga maritima for both single-stranded DNA and RNA, using heteronuclear 2D NMR spectroscopy. At "physiological" growth temperatures of TmCsp (≥ 343 K), all oligonucleotides studied have dissociation constants between 1.6 ((dT)7) and 25.2 ((dA)7) µM as determined by tryptophan fluorescence quenching. Reduction of the temperature to 303 K leads to a pronounced increase of affinity for thymidylate (dT)7 and uridylate (rU)7 heptamers with dissociation constants of 4.0 and 10.8 nM, respectively, whereas the weak binding of TmCsp to cytidylate, adenylate, and guanylate heptamers (dC)7, (dA)7, and (dT)7 is almost unaffected by temperature. The change of affinities of TmCsp for (dT)7 and (rU)7 by approximately 3 orders of magnitude shows that it represents a cold chock sensor that switches on the cold shock reaction of the cell. A temperature dependent conformational switch of the protein is required for this action. The binding epitope on TmCsp for the ssDNA and RNA heptamers is very similar and comprises ß-strands 1 and 2, the loop ß1-ß2 as well as the loops connecting ß3 with ß4 and ß4 with ß5. Besides the loop regions, surprisingly, mainly the RNA-binding motif RNP1 is involved in ssDNA and RNA binding, while only two amino acids, H28 and W29, of the postulated RNA-binding motif RNP2 interact with the uridylate and thymidylate homonucleotides, although a high affinity in the nanomolar range is achieved. This is in contrast to the binding properties of other CSPs or cold shock domains, where RNP1 as well as RNP2 are involved in binding. TmCsp takes up a unique position since it is the only one which possesses a tryptophan residue instead of a usually highly conserved phenylalanine or tyrosine residue at the end of RNP2. NMR titrations suggest that neither (dT)7 nor (rU)7 represent the full binding motif and that non-optimal intercalation of W29 into these oligonucleotides blocks the access of the RNP2 site to the DNA or RNA. NMR-experiments with (dA)7 suggest an interaction of W29 with the adenine ring. Full binding seems to require at least one single purine base well-positioned within a thymine- or uracil-rich stretch of nucleic acids.


Assuntos
Proteínas de Bactérias/química , Proteínas e Peptídeos de Choque Frio/química , Proteínas de Ligação a DNA/química , Epitopos/química , Proteínas de Ligação a RNA/química , Thermotoga maritima/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas e Peptídeos de Choque Frio/genética , Proteínas de Ligação a DNA/genética , Epitopos/genética , Proteínas de Ligação a RNA/genética , Thermotoga maritima/genética
19.
Int J Mol Sci ; 21(20)2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053818

RESUMO

Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.


Assuntos
Arginina/química , Arginina/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Transporte/genética , Hiperargininemia/diagnóstico , Hiperargininemia/etiologia , Hiperargininemia/metabolismo , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Thermotoga maritima/genética
20.
Sci Rep ; 10(1): 15404, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958806

RESUMO

The large catalytic subunit of acetohydroxyacid synthase (AHAS, EC 2.2.1.6) of Thermotoga maritima (TmcAHAS) was prepared in this study. It possesses high specific activity and excellent stability. The protein and a whole cell catalyst overexpressing the protein were applied to the preparation of α-hydroxyketones including acetoin (AC), 3-hydroxy-2-pentanone (HP), and (R)-phenylacetylcarbinol (R-PAC). The results show that AC and HP could be produced in high yields (84% and 62%, respectively), while R-PAC could be synthesized in a high yield (about 78%) with an R/S ratio of 9:1. Therefore, TmcAHAS and the whole cell catalyst overexpressing the protein could be practically useful bio-catalysts in the preparation of α-hydroxyketones including AC, HP, and R-PAC. To the best of our knowledge, this is the first time that bacterial AHAS was used as a catalyst to prepare HP with a good yield, and also the first time that TmcAHAS was employed to synthesize AC and R-PAC.


Assuntos
Acetolactato Sintase/isolamento & purificação , Acetolactato Sintase/metabolismo , Catálise , Domínio Catalítico , Cetonas , Proteínas Recombinantes/isolamento & purificação , Thermotoga maritima/metabolismo
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