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1.
Phys Chem Chem Phys ; 23(39): 22692-22702, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34605505

RESUMO

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.


Assuntos
Dinitrocresóis/metabolismo , Simulação de Dinâmica Molecular , Timidilato Sintase/metabolismo , Biocatálise , Dinitrocresóis/química , Estrutura Molecular , Oxirredução , Thermotoga maritima/enzimologia , Timidilato Sintase/química
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.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
Phys Chem Chem Phys ; 22(33): 18361-18373, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32789320

RESUMO

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.


Assuntos
Proteínas de Escherichia coli/química , Tetra-Hidrofolato Desidrogenase/química , Catálise , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Maleabilidade , Conformação Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Desdobramento de Proteína , Thermotoga maritima/enzimologia , Temperatura de Transição
14.
Enzyme Microb Technol ; 139: 109579, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32732029

RESUMO

Endo-ß-1,4-xylanase from Thermotoga maritima, TmxB, is an industrially attractive enzyme due to its extreme thermostability. To improve its application value, four variants were designed on the basis of multiple sequence and three-dimensional structure alignments. Wild-type TmxB (wt-TmxB) and its mutants were produced via a Pichia pastoris expression system. Among four single-site mutants, the tyrosine substitution of a threonine residue (T74Y) at putative -3/-4 subsite led to a 1.3-fold increase in specific activity at 40 °C - 100 °C and pH 5 for 5 min, with beechwood xylan as the substrate. T74Y had an improved catalytic efficiency (kcat/Km), being 1.6 times that of wt-TmxB. Variants DY (two amino acid insertions) and N68Q displayed a slight increase (1.2 fold) and dramatic decline (1.7 fold) in catalytic efficiency, respectively. Mutant E67Y was totally inactive under all test conditions. Structural modeling and docking simulation elucidated structural insights into the molecular mechanism of activity changes for these TmxB variants. This study helps in further understanding the roles of the non-catalytic amino acids at the glycone subsites of xylanases from glycoside hydrolase family 10.


Assuntos
Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Mutagênese Sítio-Dirigida , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , Domínio Catalítico , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Modelos Estruturais , Simulação de Acoplamento Molecular , Saccharomycetales/genética , Saccharomycetales/metabolismo , Especificidade por Substrato
15.
Commun Biol ; 3(1): 431, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770029

RESUMO

rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Fermentação , Sódio/metabolismo , Thermotoga maritima/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Dicicloexilcarbodi-Imida/farmacologia , Fermentação/efeitos dos fármacos , Ferredoxinas/metabolismo , Glucose/metabolismo , Hidrólise , Transporte de Íons/efeitos dos fármacos , Ionóforos/farmacologia , Lipossomos , Modelos Biológicos , Oxirredutases/metabolismo , Subunidades Proteicas/isolamento & purificação , ATPases Translocadoras de Prótons/isolamento & purificação , ATPases Translocadoras de Prótons/metabolismo , Thermotoga maritima/efeitos dos fármacos
16.
Biochemistry ; 59(29): 2729-2742, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32633500

RESUMO

Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure analysis employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ∼18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover number. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissociation constant for its substrate, glutamine. In addition, AzoF improved the apparent binding of a substrate analogue at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.


Assuntos
Aminoidrolases/química , Thermotoga maritima/enzimologia , Regulação Alostérica , Aminoidrolases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Glutamina/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica , Thermotoga maritima/química , Thermotoga maritima/metabolismo
17.
Proteins ; 88(12): 1639-1647, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32673419

RESUMO

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.


Assuntos
Aminopeptidases/química , Aminopeptidases/metabolismo , Ácido Aspártico/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Thermotoga maritima/enzimologia , Aminopeptidases/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
18.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478746

RESUMO

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.


Assuntos
Aminopeptidases/química , Cristalografia por Raios X , Multimerização Proteica , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Aminopeptidases/isolamento & purificação , Aminopeptidases/metabolismo , Cromatografia em Gel , Cristalização , Recombinação Homóloga , Modelos Moleculares
19.
Biotechnol Prog ; 36(6): e3033, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32506832

RESUMO

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d-galactose to d-tagatose at high temperature. l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His)6 -tagged protein, immobilized on a copper-chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d-galactose to d-tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support.


Assuntos
Aldose-Cetose Isomerases/química , Enzimas Imobilizadas/química , Galactose/química , Hexoses/biossíntese , Estabilidade Enzimática/genética , Enzimas/química , Enzimas/farmacologia , Hexoses/química , Thermotoga maritima/enzimologia
20.
J Mol Biol ; 432(16): 4762-4771, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32592697

RESUMO

Reverse gyrase is a unique type I topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. Supercoiling is the result of a functional cooperation of the N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain is a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. The isolated helicase domain as well as full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain with little conservation in sequence and length, has been proposed to coordinate events in the helicase domain with strand passage by the topoisomerase domain. Latch deletions lead to a reduction in or complete loss of supercoiling activity. Here we show that the latch consists of two functional parts, a globular domain that is dispensable for DNA supercoiling and a ß-hairpin that connects the globular domain to the helicase domain and is required for supercoiling activity. The ß-hairpin thus constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain.


Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/metabolismo , Thermotoga maritima/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Topoisomerases Tipo I/genética , DNA Bacteriano/metabolismo , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Deleção de Sequência , Thermotoga maritima/química , Thermotoga maritima/genética
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