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1.
Sci Rep ; 11(1): 16430, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385527

RESUMO

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
2.
DNA Repair (Amst) ; 78: 45-59, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959406

RESUMO

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase that removes oxidized pyrimidines from DNA. The genome of Deinococcus radiodurans encodes for an unusually high number of DNA glycosylases, including three EndoIII enzymes (drEndoIII1-3). Here, we compare the properties of these enzymes to those of their well-studied homologues from E. coli and human. Our biochemical and mutational data, reinforced by MD simulations of EndoIII-DNA complexes, reveal that drEndoIII2 exhibits a broad substrate specificity and a catalytic efficiency surpassing that of its counterparts. In contrast, drEndoIII1 has much weaker and uncoupled DNA glycosylase and AP-lyase activities, a characteristic feature of eukaryotic DNA glycosylases, and was found to present a relatively robust activity on single-stranded DNA substrates. To our knowledge, this is the first report of such an activity for an EndoIII. In the case of drEndoIII3, no catalytic activity could be detected, but its ability to specifically recognize lesion-containing DNA using a largely rearranged substrate binding pocket suggests that it may play an alternative role in genome maintenance. Overall, these findings reveal that D. radiodurans possesses a unique set of DNA repair enzymes, including three non-redundant EndoIII variants with distinct properties and complementary activities, which together contribute to genome maintenance in this bacterium.


Assuntos
Reparo do DNA , DNA Complementar/genética , Deinococcus/enzimologia , Deinococcus/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Mutação , Biocatálise , DNA Complementar/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Pirimidinas/metabolismo , Especificidade por Substrato
3.
J Biol Chem ; 294(1): 157-167, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30420426

RESUMO

Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.


Assuntos
Organismos Aquáticos/química , Proteínas de Bactérias/química , Shewanella/química , Sideróforos , Organismos Aquáticos/genética , Proteínas de Bactérias/genética , Flavina-Adenina Dinucleotídeo/química , NADP/química , Domínios Proteicos , Shewanella/genética
4.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 7): 419-424, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29969105

RESUMO

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and small- and medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ∼10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 Šresolution. The crystals belonged to the trigonal space group P31 or P32, with unit-cell parameters a = b = 64.13, c = 111.32 Å, and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ∼30% sequence identity at ∼2 Šresolution as templates are ongoing.


Assuntos
Clonagem Molecular/métodos , Deinococcus/enzimologia , Deinococcus/genética , Peroxidase/química , Peroxidase/genética , Sequência de Aminoácidos , Cristalização/métodos , Regulação Bacteriana da Expressão Gênica , Peroxidase/biossíntese , Difração de Raios X/métodos
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 188: 149-154, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28709140

RESUMO

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex.


Assuntos
Biocatálise , Eletroquímica/métodos , Endonucleases/química , Endonucleases/metabolismo , Mutação/genética , Aminoácidos/química , Sequência Conservada , Deinococcus , Enzimas Imobilizadas/metabolismo , Modelos Moleculares , Oxirredução , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
7.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 9): 667-71, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27599855

RESUMO

Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Šresolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, ß = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Šresolution SIP structures with ∼30% sequence identity as templates are ongoing.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Shewanella/química , Sideróforos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Shewanella/metabolismo , Sideróforos/metabolismo , Difração de Raios X
8.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 10): 2137-49, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26457437

RESUMO

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.


Assuntos
DNA/metabolismo , Deinococcus/enzimologia , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , DNA/química , Deinococcus/química , Deinococcus/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência
9.
J Struct Biol ; 191(2): 87-99, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26172070

RESUMO

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.


Assuntos
Proteínas de Bactérias/química , Deinococcus/enzimologia , Desoxirribonuclease (Dímero de Pirimidina)/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Relação Estrutura-Atividade
10.
BMC Struct Biol ; 15: 5, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25886944

RESUMO

BACKGROUND: Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can tolerate radiation doses up to 5,000 Grays without losing viability. We are studying the role of DNA repair and replication proteins for this unusual phenotype by a structural biology approach. The DNA polymerase III ß subunit (ß-clamp) acts as a sliding clamp on DNA, promoting the binding and processivity of many DNA-acting proteins, and here we report the crystal structure of D. radiodurans ß-clamp (Drß-clamp) at 2.0 Å resolution. RESULTS: The sequence verification process revealed that at the time of the study the gene encoding Drß-clamp was wrongly annotated in the genome database, encoding a protein of 393 instead of 362 amino acids. The short protein was successfully expressed, purified and used for crystallisation purposes in complex with Cy5-labeled DNA. The structure, which was obtained from blue crystals, shows a typical ring-shaped bacterial ß-clamp formed of two monomers, each with three domains of identical topology, but with no visible DNA in electron density. A visualisation of the electrostatic surface potential reveals a highly negatively charged outer surface while the inner surface and the dimer forming interface have a more even charge distribution. CONCLUSIONS: The structure of Drß-clamp was determined to 2.0 Å resolution and shows an evenly distributed electrostatic surface charge on the DNA interacting side. We hypothesise that this charge distribution may facilitate efficient movement on encircled DNA and help ensure efficient DNA metabolism in D. radiodurans upon exposure to high doses of ionizing irradiation or desiccation.


Assuntos
Proteínas de Bactérias/química , DNA Polimerase III/química , Deinococcus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Bacteriano/metabolismo , Deinococcus/química , Deinococcus/enzimologia , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade Estática
11.
FEBS Open Bio ; 5: 107-16, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25737836

RESUMO

Upon infection by pathogenic bacteria, production of reactive oxygen species (ROS) is part of the host organism's first line of defence. ROS damage a number of macromolecules, and in order to withstand such a harsh environment, the bacteria need to have well-functioning ROS scavenging and repair systems. Herein, MutT is an important nucleotide-pool sanitization enzyme, which degrades 8-oxo-dGTP and thus prevents it from being incorporated into DNA. In this context, we have performed a comparative biochemical and structural analysis of MutT from the fish pathogen Aliivibrio salmonicida (AsMutT) and the human pathogen Vibrio cholerae (VcMutT), in order to analyse their function as nucleotide sanitization enzymes and also determine possible cold-adapted properties of AsMutT. The biochemical characterisation revealed that both enzymes possess activity towards the 8-oxo-dGTP substrate, and that AsMutT has a higher catalytic efficiency than VcMutT at all temperatures studied. Calculations based on the biochemical data also revealed a lower activation energy (E a) for AsMutT compared to VcMutT, and differential scanning calorimetry experiments showed that AsMutT displayed an unexpected higher melting temperature (T m) value than VcMutT. A comparative analysis of the crystal structure of VcMutT, determined to 2.42 Å resolution, and homology models of AsMutT indicate that three unique Gly residues in loops of VcMutT, and additional long range ion-pairs in AsMutT could explain the difference in temperature stability of the two enzymes. We conclude that AsMutT is a stable, cold-active enzyme with high catalytic efficiency and reduced E a, compared to the mesophilic VcMutT.

12.
Chem Commun (Camb) ; 51(15): 3255-7, 2015 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-25608720

RESUMO

Surface enhanced vibrational spectro-electrochemistry of endonuclease III provides direct evidence that the [4Fe-4S] cluster is responsible for the enzyme redox activity, and that this process is not exclusively DNA-mediated, as currently proposed. We report the first surface enhanced resonance Raman spectrum of a [4Fe-4S](2+) cluster containing enzyme.


Assuntos
Proteínas de Bactérias/química , Endonucleases/química , Enzimas Imobilizadas/química , Deinococcus/enzimologia , Eletroquímica , Ferro/química , Oxirredução , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Enxofre/química
13.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 12): 1688-92, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25484227

RESUMO

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Šresolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, ß = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, ß = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.


Assuntos
Deinococcus/enzimologia , Endonucleases/metabolismo , Cristalização , Cristalografia por Raios X , Endonucleases/química
14.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 8): 2093-100, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25084329

RESUMO

Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme-substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG-Ugi was determined to a resolution of 1.9 Šwith eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG-Ugi complex with previously determined structures of UNG-Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (Kb) which is one order of magnitude larger than that for hUNG-Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein-Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.


Assuntos
Inibidores Enzimáticos/farmacologia , Uracila-DNA Glicosidase/metabolismo , Animais , Biofísica , Gadus morhua , Humanos , Conformação Proteica , Termodinâmica , Uracila-DNA Glicosidase/antagonistas & inibidores , Uracila-DNA Glicosidase/química
15.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 6): 703-12, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22683793

RESUMO

3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/ß domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket.


Assuntos
DNA Glicosilases/química , Deinococcus/enzimologia , Sequência de Aminoácidos , DNA/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Especificidade por Substrato
16.
Artigo em Inglês | MEDLINE | ID: mdl-21959147

RESUMO

Uracil-DNA N-glycosylase (UNG; EC 3.2.2.27) from Atlantic cod (cUNG) possesses cold adapted features like increased catalytic efficiency and reduced temperature optimum for activity compared to its warm-adapted homologue human UNG (hUNG). Here, we present the first thermal stability analysis of cUNG and hUNG by differential scanning calorimetry (DSC), and the results showed that cUNG is less stable than hUNG and unfolds at a melting temperature (T(m)) 9° lower than its warm-adapted homologue. In addition, an ion-pair (D183-K302) suggested to be crucial for global stability of hUNG was investigated by biochemical characterization and DSC of four mutants (cUNG G183D and cUNG G183D-R302K, hUNG D183G and hUNG D183G-K302R). The hUNG mutants with an expected disruption of the ion-pair showed a slight increase in stability with concomitant reduction in the enzyme activity, while the apparent introduction of the ion-pair in cUNG caused a reduction in the enzyme activity but no increase in stability. Because the mutants did not behave as expected, the phenomenon was further investigated by crystal structure determination. Indeed, the crystal structure of the hUNG D183G-K302R mutant revealed that compensating interactions for the loss of the ion-pair were generated close to and in regions distant from the mutation site. In conclusion, the reduced stability of cUNG supports the suggested requirement of a flexible structure for improved activity at low temperatures. Furthermore, the lack of a direct correlation between enzyme activity and global stability of the mutants supports the significance of distributing locally flexible and/or rigid regions for modulation of enzyme activity.


Assuntos
Adaptação Fisiológica , Temperatura Baixa , Gadus morhua/metabolismo , Desdobramento de Proteína , Temperatura , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Substituição de Aminoácidos/genética , Animais , Oceano Atlântico , Biocatálise , Tampões (Química) , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Estabilidade Enzimática , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Íons , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Conformação Proteica , Uracila-DNA Glicosidase/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-20124707

RESUMO

The crystal structure of Vibrio cholerae uracil-DNA N-glycosylase (vcUNG) has been determined to 1.5 A resolution. Based on this structure, a homology model of Aliivibrio salmonicida uracil-DNA N-glycosylase (asUNG) was built. A previous study demonstrated that asUNG possesses typical cold-adapted features compared with vcUNG, such as a higher catalytic efficiency owing to increased substrate affinity. Specific amino-acid substitutions in asUNG were suggested to be responsible for the increased substrate affinity and the elevated catalytic efficiency by increasing the positive surface charge in the DNA-binding region. The temperature adaptation of these enzymes has been investigated using structural and mutational analyses, in which mutations of vcUNG demonstrated an increased substrate affinity that more resembled that of asUNG. Visualization of surface potentials revealed a more positive potential for asUNG compared with vcUNG; a modelled double mutant of vcUNG had a potential around the substrate-binding region that was more like that of asUNG, thus rationalizing the results obtained from the kinetic studies.


Assuntos
Mutação , Uracila-DNA Glicosidase/química , Vibrio cholerae/enzimologia , Adaptação Biológica , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Temperatura , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
18.
Acta Crystallogr D Biol Crystallogr ; 64(Pt 4): 368-76, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18391403

RESUMO

The crystal structure of the periplasmic/extracellular endonuclease I from Vibrio salmonicida has been solved to 1.5 A resolution and, in comparison to the corresponding endonucleases from V. cholerae and V. vulnificus, serves as a model system for the investigation of the structural determinants involved in the temperature and NaCl adaptation of this enzyme class. The overall fold of the three enzymes is essentially similar, but the V. salmonicida endonuclease displays a significantly more positive surface potential than the other two enzymes owing to the presence of ten more Lys residues. However, if the optimum salt concentrations for the V. salmonicida and V. cholerae enzymes are taken into consideration in the electrostatic surface-potential calculation, the potentials of the two enzymes become surprisingly similar. The higher number of basic residues in the V. salmonicida protein is therefore likely to be a result, at least in part, of adaptation to the more saline habitat of V. salmonicida (seawater) than V. cholerae (brackish water). The hydrophobic core of all three enzymes is almost identical, but the V. salmonicida endonuclease has a slightly lower number of internal hydrogen bonds. This, together with repulsive forces between the basic residues on the protein surface of V. salmonicida endonuclease I and differences in the distribution of salt bridges, probably results in higher flexibility of regions of the V. salmonicida protein. This is likely to influence both the catalytic activity and the stability of the protein.


Assuntos
Adaptação Fisiológica/fisiologia , Aliivibrio salmonicida/enzimologia , Aliivibrio salmonicida/fisiologia , Temperatura Baixa , Desoxirribonuclease I/química , Desoxirribonuclease I/fisiologia , Sequência de Aminoácidos , Cristalografia por Raios X , Interpretação Estatística de Dados , Focalização Isoelétrica , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Cloreto de Sódio
19.
J Biol Chem ; 282(30): 21973-86, 2007 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-17537732

RESUMO

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH(4)) as cofactor and at 25 degrees C (15 degrees C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S(0.5)](L-Phe) (= 1.1 +/- 0.1 mm) and K(m)(BH(4))(= 0.3 +/- 0.1 mm), as well as high catalytic efficiency at 10 degrees C. However, the half-inactivation and denaturation temperature is only slightly lowered (T(m) approximately 52 degrees C; where T(m) is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH(4) (Gly(87)/Phe(88)/Gly(89)) and l-Phe (Tyr(114)-Pro(118)). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.


Assuntos
Alteromonadaceae/enzimologia , Alteromonadaceae/genética , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/metabolismo , Aclimatação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Estabilidade Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos , Genes Precoces , Ligação de Hidrogênio , Dados de Sequência Molecular , Fenilalanina Hidroxilase/genética , Conformação Proteica
20.
Comput Biol Chem ; 31(3): 163-72, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17500034

RESUMO

The periplasmic/extracellular bacterial enzyme endonuclease I was chosen as a model system to identify features that might be responsible for temperature- and salt adaptation. A statistical study of amino acid sequence properties belonging to endonuclease I enzymes from three mesophilic habitats (non-marine, brackish water and marine), and three marine temperature groups (psychrophile, intermediate and mesophile) has been conducted. Ten new endonuclease I genes have been sequenced in order to increase the sample size. A bioinformatical method of property dependent statistical analysis of alignments has been applied. To our knowledge this is the first time these methods have been used in order to investigate environmental adaptation of enzymes. Adaptation to low temperature seems to involve increased surface isoelectric point and hydrophobicity in contrast to salt adaptation in which the isoelectric point and hydrophobicity at the surface decreases. Redistribution of charge and hydrophobicity might be the most important signature for cold adaptation and salt adaptation of this enzyme class. The results indicate that general trends of adaptation are possible to elucidate from the amino acid sequences. Also in this paper a new scale of stratified B-factors, derived from the Protein Data Bank, is presented.


Assuntos
Adaptação Fisiológica/genética , Bactérias/genética , Desoxirribonuclease I/genética , Alinhamento de Sequência/métodos , Substituição de Aminoácidos , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Temperatura Baixa , Biologia Computacional/métodos , Bases de Dados de Proteínas , Desoxirribonuclease I/química , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Ponto Isoelétrico , Concentração Osmolar , Conformação Proteica , Estrutura Secundária de Proteína , Sais/farmacologia , Análise de Sequência de DNA , Propriedades de Superfície , Temperatura
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