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1.
Gene ; 720: 144082, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476406

RESUMO

The enzyme ß-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115-147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Proteínas de Bactérias/química , Isoenzimas/química , Proteínas Mutantes/química , Mutação , Mycobacterium tuberculosis/enzimologia , Tuberculose/microbiologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Proteínas de Bactérias/genética , Isoenzimas/genética , Simulação de Dinâmica Molecular , Proteínas Mutantes/genética , Conformação Proteica , Tuberculose/genética , Tuberculose/metabolismo
2.
Phys Chem Chem Phys ; 21(32): 17950-17958, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31384849

RESUMO

The A. aeolicus intrinsically disordered protein FlgM has four well-defined α-helices when bound to σ28, but in water FlgM undergoes a change in tertiary structure. In this work, we investigate the structure of FlgM in aqueous solutions of the ionic liquid [C4mpy][Tf2N]. We find that FlgM is induced to fold by the addition of the ionic liquid, achieving average α-helicity values similar to the bound state. Analysis of secondary structure reveals significant similarity with the bound state, but the tertiary structure is found to be more compact. Interestingly, the ionic liquid is not homogeneously dispersed in the water, but instead aggregates near the protein. Separate simulations of aqueous ionic liquid do not show ion clustering, which suggests that FlgM stabilizes ionic liquid aggregation.


Assuntos
Proteínas de Bactérias/química , Imidas/química , Proteínas Intrinsicamente Desordenadas/química , Líquidos Iônicos/química , Modelos Moleculares , Pirrolidinas/química , Bases de Dados de Proteínas , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Termodinâmica , Água
3.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
4.
J Agric Food Chem ; 67(37): 10392-10400, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31461615

RESUMO

The specificity of fructooligosaccharides as prebiotics depends on their size and structure, which in turn depend on their origin or the synthesis procedure. In this work we describe the application of an inulosucrase (IslA) from Leuconostoc citreum CW28 to produce high molecular weight inulin from sucrose alongside a commercial endoinulinase (Novozym 960) produced by Aspergillus niger for a simultaneous or sequential reaction to synthesize fructooligosaccharides (FOS). The simultaneous reaction resulted in a higher substrate conversion and a wide diversity of FOS when compared to the sequential reaction. A shotgun MS analysis of the commercial endoinulinase preparation surprisingly revealed an additional enzymatic activity: a fructosyltransferase, responsible for the synthesis of FOS from sucrose. Consequentially, the range of FOS obtained in reactions combining inulosucrase from Ln. citreum with the fructosyltransferase and endoinulinase from A. niger with sucrose as substrate may be extended and regulated.


Assuntos
Proteínas de Bactérias/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Hexosiltransferases/química , Inulina/química , Leuconostoc/enzimologia , Oligossacarídeos/química , Aspergillus niger/enzimologia , Biocatálise , Sacarose/química
5.
Inorg Chem ; 58(17): 11351-11363, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31433627

RESUMO

Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from Listeria innocua to structurally characterize these steps for Co2+, Zn2+, and La3+ ions. The structures reveal that differences in their ion coordination chemistry determine alternative metal ion-binding sites on the areas of the surface surrounding the translocation pore that captures nine La3+, three Co2+, or three Zn2+ ions as aquo clusters and passes them on for translocation. Inside these pores, ion-selective conformational changes at key residues occur before a gating residue to actively move ions through the constriction zone. Ions upstream of the Asp130 gate residue are typically hydrated, while ions downstream directly interact with the protein matrix. Inside the cavity, ions move along negatively charged residues to the ferroxidase center, where seven main residues adapt to the three different ions by dynamically changing their conformations. In total, we observed more than 20 metal-binding sites per Dps monomer, which clearly highlights the metal-binding capacity of this protein family. Collectively, our results provide a detailed structural description of the preparative steps for amino acid-assisted biomineralization in Dps proteins, demonstrating unexpected protein matrix plasticity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Listeria/química , Metais Pesados/química , Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/biossíntese , Modelos Moleculares , Eletricidade Estática
6.
J Agric Food Chem ; 67(35): 9868-9876, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389242

RESUMO

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.


Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Thermus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Alinhamento de Sequência , Thermus/química , Thermus/genética
7.
J Chem Theory Comput ; 15(8): 4535-4546, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31264415

RESUMO

Anabaena Sensory Rhodopsin (ASR), a microbial photoactive protein featuring the retinal chromophore in two different conformations, exhibits a pH-dependent electronic absorption spectrum. Using the recently developed CpHMD-then-QM/MM multiscale protocol applied to ASR embedded in a membrane model, the pH-induced changes in its maximum absorption wavelength have been reproduced and analyzed. While the acidic tiny red-shift is essentially correlated with the deprotonation of an aspartic acid located on the ASR extracellular side, the larger blue-shift experimentally reported at pH values larger than 5 involves a cluster of titrating residues sitting on the cytoplasmic side. The ASR pH-dependent spectrum is the consequence of the competitive stabilization of retinal ground and excited states by the protein electrostatic potential.


Assuntos
Aminoácidos/química , Anabaena/química , Proteínas de Bactérias/química , Nostoc/química , Rodopsinas Sensoriais/química , Aminoácidos/análise , Ácido Aspártico/análise , Ácido Aspártico/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Prótons , Espectrofotometria , Eletricidade Estática
8.
J Agric Food Chem ; 67(32): 8896-8904, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339308

RESUMO

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.


Assuntos
Aedes/efeitos dos fármacos , Aedes/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/genética , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos
9.
J Agric Food Chem ; 67(31): 8548-8558, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31266305

RESUMO

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.


Assuntos
Proteínas de Bactérias/química , Esterases/química , Ésteres/química , Gordonia (Bactéria)/enzimologia , Ácidos Ftálicos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Biodegradação Ambiental , Dietilexilftalato/química , Dietilexilftalato/metabolismo , Esterases/genética , Esterases/metabolismo , Ésteres/metabolismo , Gordonia (Bactéria)/genética , Gordonia (Bactéria)/isolamento & purificação , Gordonia (Bactéria)/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Alinhamento de Sequência , Microbiologia do Solo
10.
J Agric Food Chem ; 67(31): 8527-8535, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298526

RESUMO

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.


Assuntos
Proteínas de Bactérias/química , Corynebacterium glutamicum/enzimologia , Cetol-Ácido Redutoisomerase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Cristalografia por Raios X , Cetol-Ácido Redutoisomerase/genética , Cetol-Ácido Redutoisomerase/metabolismo , Metais/química , Metais/metabolismo , Simulação de Acoplamento Molecular , NADP/química , NADP/metabolismo
11.
Nat Commun ; 10(1): 2917, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266949

RESUMO

Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design.


Assuntos
Antibacterianos/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Produtos Biológicos/química , Inibidores Enzimáticos/química , Nucleosídeos/antagonistas & inibidores , Transferases/química , Aminoglicosídeos/química , Arginina/análogos & derivados , Arginina/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo
12.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
13.
Phys Chem Chem Phys ; 21(30): 16515-16525, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31298238

RESUMO

Isoniazid (INH) is converted into isonicotinyl radical through its interaction with the catalase-peroxidase (katG) enzyme present in the cells of Mycobacterium tuberculosis (M. tb.), the bacteria that causes the tuberculosis disease. This process is important because resistance of M. tb. cells to INH treatment has been associated with the failure of completion of this process. However, this process is poorly understood and there are a variety of conflicting theories about the details of the mechanism of this process. One theory suggests that INH binds to katG and transfers a single electron to the heme while the heme is in its two electron oxidized state, compound I [Fe(iv)Por˙+] (CpdI). In this study, DFT calculations at the UB3LYP/6-31g(d)-LANL2DZ level of theory are used to study the M. tb. katG CpdI molecule. Different models of the M. tb. CpdI molecule were prepared and the calculations revealed the impact of Trp321 on the electronic properties of the heme. Without Trp321 the heme assumed a triradical state with single electrons on the πxy and πyz orbitals of Fe and another on the a2u orbital of the porphyrin ring that can either be coupled with the first two, to assume a quartet state, or decoupled to form a doublet state. With Trp321, however, a transfer of an electron from the πTrp orbital to a2u porphyrin orbital leads to loss of radical character of the porphyrin and leaves the Trp321 group with a radical character. INH was observed to have strong interaction with CpdI and the absence of Trp321 significantly decreased the binding energy by 2 kcal mol-1 explaining the importance of Trp321 in the binding of INH. The results in this study show the importance of Trp321 in the binding of INH and its effect on its electronic properties, which is consistent with previous experimental findings that mutation of Trp321 results in an increase in drug resistance.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catalase/química , Catalase/metabolismo , Teoria da Densidade Funcional , Isoniazida/metabolismo , Antituberculosos/metabolismo
14.
J Agric Food Chem ; 67(29): 8177-8185, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31290662

RESUMO

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, and is therefore essential for trehalose production. Consequently, dissecting the catalytic mechanism of TreS is important for enzyme optimization and industrial applications. TreS from Thermobaculum terrenum (TtTreS) is a thermostable enzyme. Here, we studied the composition of the TtTreS active site through computer calculation and enzyme analysis. The results were consistent with a two-step double-displacement mechanism, similar to that of glycoside hydrolase 13 family enzymes. However, our data suggested that glucose rotation, following breakage of the α-1,4 glycosidic bond, is a key factor determining the reaction direction and conversion rate. The N246 residue plays an important role in glucose rotation. Moreover, we established a saturated mutation model for the nonconserved amino acids around the substrate gateway domain. Finally, four TtTreS mutants (K136T, Y137D, K138N, and D139S) resulted in improved trehalose yield compared to that of the wild-type enzyme.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Glucosiltransferases/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Biologia Computacional , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Especificidade por Substrato
15.
Inorg Chem ; 58(16): 11091-11099, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31353893

RESUMO

Several bacterial species have evolutionary developed protein systems specialized in the control of intracellular gold ion concentration. In order to prevent the detrimental consequences that may be induced even at very low concentrations, bacteria such as Salmonella enterica and Cupriavidus metallidurans utilize Au-specific merR-type transcriptional regulators that detect these toxic ions and control the expression of specific resistance factors. Among these highly specialized proteins, golB has been investigated in depth, and X-ray structures of both apo and Au(I)-bound golB have been recently reported. Here, the binding of Au(I) at golB was investigated by means of multilevel computational approaches. Molecular dynamics simulations evidenced how conformations amenable for the Au(I) chelation through the Cys-XX-Cys motif on helix 1 are extensively sampled in the phase space of apo-golB. Hybrid QM/MM calculations on metal-bound structures of golB also allowed to characterize the most probable protonation state for gold binding motif and to assess the structural features mostly influencing the Au(I) coordination in this protein. Consistently with experimental evidence, we found that golB may control its Au(I) affinity by conformational changes that affect the distance between Cys10 and Cys13, thus being able to switch between the Au(I) sequestration/release-prone states in response to external stimuli. The protein structure enveloping the metal binding motif favors the thiol-thiolate protonation state of Au(I)-golB, thus probably enhancing the binding selectivity for Au(I) compared to other cations.


Assuntos
Proteínas de Bactérias/química , Ouro/química , Metaloproteínas/química , Simulação de Dinâmica Molecular , Teoria Quântica , Cupriavidus/química , Salmonella enterica/química
16.
J Biochem ; 166(2): 121-127, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31340005

RESUMO

Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging.


Assuntos
Proteínas de Bactérias/química , Corantes Fluorescentes/química , Imagem Óptica , Fotorreceptores Microbianos/química , Células HEK293 , Humanos , Estrutura Molecular , Processos Fotoquímicos
17.
Adv Mater ; 31(33): e1902462, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31265196

RESUMO

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Cisteína Endopeptidases/química , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/química , Proteínas Recombinantes/química , Aminoaciltransferases/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Cisteína Endopeptidases/administração & dosagem , Dissulfetos/química , Liberação Controlada de Fármacos , Humanos , Luz , Oxirredução , Peptídeos/química , Fotólise , Polietilenoglicóis/química , Proteínas Recombinantes/administração & dosagem , Staphylococcus aureus/enzimologia
18.
Chemistry ; 25(51): 11837-11841, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31310409

RESUMO

Bacterial production of ß-lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA-48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post-translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19 F NMR spectroscopy to monitor the carbamylation state of 19 F-labelled OXA-48. This method was used to investigate the interactions of OXA-48 with clinically used serine ß-lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19 F-labelled OXA-48 provide a structural rationale for the sensitivity of the 19 F label to active site interactions. The overall results demonstrate the use of 19 F NMR to monitor reversible covalent post-translational modifications.


Assuntos
Compostos Azabicíclicos/química , Proteínas de Bactérias/química , Radioisótopos de Flúor/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/farmacologia , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância Magnética , Carbamilação de Proteínas , Processamento de Proteína Pós-Traducional , Inibidores de beta-Lactamases/química , beta-Lactamases/química , beta-Lactamases/metabolismo
20.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 564-577, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205019

RESUMO

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.


Assuntos
Proteínas de Bactérias/química , N-Acilneuraminato Citidililtransferase/química , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferase/farmacologia , N-Acilneuraminato Citidililtransferase/fisiologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Ácidos Siálicos/metabolismo
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