RESUMO
Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailed mechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.
Assuntos
Deinococcus/enzimologia , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Cristalografia por Raios X , Cinética , Ligantes , Fosforilação , Conformação Proteica , Especificidade por SubstratoRESUMO
During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.
Assuntos
Adaptação Biológica/fisiologia , Francisella tularensis/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Ilhas Genômicas/genética , Transcriptoma/fisiologia , Virulência/fisiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Genes Reguladores/genética , Genes Reguladores/fisiologia , Ilhas Genômicas/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Proteoma/fisiologia , Análise de Sequência de RNA , Serina/análogos & derivados , Serina/toxicidade , Estresse Fisiológico , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Transcriptoma/genética , Virulência/genéticaRESUMO
Hydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. The most active subclass, the [FeFe]-hydrogenases, is dependent on a metallocofactor, the H cluster, that consists of a two iron subcluster ([2Fe]H) bridging to a classical cubane cluster ([4Fe-4S]H). The ligands coordinating to the diiron subcluster include an azadithiolate, three carbon monoxides, and two cyanides. To assemble this complex cofactor, three maturase enzymes, HydG, HydE and HydF are required. The biosynthesis of the diatomic ligands proceeds by an unusual fragmentation mechanism, and structural studies in combination with spectroscopic analysis have started to provide insights into the HydG mediated assembly of a [2Fe]H subcluster precursor.
Assuntos
Coenzimas/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Coenzimas/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismoRESUMO
The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel ß-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.
Assuntos
Proteínas de Bactérias/química , Francisella tularensis/enzimologia , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Animais , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Francisella tularensis/genética , Camundongos , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Estrutura Secundária de ProteínaRESUMO
The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 µM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.
Assuntos
Francisella tularensis/enzimologia , Ligases/metabolismo , Ribossomos/enzimologia , Regulação Alostérica/genética , Escherichia coli/enzimologia , Cinética , Ligases/genéticaRESUMO
Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (ßα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a µ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hidrogênio/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Domínio Catalítico , Modelos Moleculares , Conformação Proteica , Tirosina/químicaRESUMO
Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enxofre/metabolismo , Sulfurtransferases/química , Sulfurtransferases/metabolismo , Sítios de Ligação/fisiologia , Cristalização , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.
Assuntos
Clostridium acetobutylicum/enzimologia , Hidrogenase/química , Proteínas Ferro-Enxofre/química , S-Adenosilmetionina/química , Catálise , Clostridium acetobutylicum/genética , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/genética , Proteínas Ferro-Enxofre/genética , Cinética , Mutagênese Sítio-Dirigida , Estrutura Terciária de ProteínaRESUMO
The 'radical S-adenosyl-L-methionine (AdoMet)' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (â¼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.
Assuntos
Cisteína/metabolismo , Proteínas de Escherichia coli/biossíntese , Radicais Livres/metabolismo , Metiltransferases/biossíntese , S-Adenosilmetionina/metabolismo , Desoxiadenosinas/biossíntese , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/genética , Ligantes , Metilação , Metiltransferases/genética , Ligação Proteica , Proteínas RecombinantesRESUMO
A series of bisubstrate inhibitors for DNA N6 adenine methyltransferase (Dam) have been synthesized by linking an amine analogue of S-adenosylmethionine to an aryl moiety designed to probe the binding pocket of the DNA adenine base. An initial structure-activity relationship study has identified substituents that increase inhibitor potency to the â¼10 µM range and improve selectivity against the human cytosine methyltransferase Dnmt1.
Assuntos
Inibidores Enzimáticos/síntese química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/antagonistas & inibidores , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , S-Adenosilmetionina , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A large superfamily of enzymes have been identified that make use of radical intermediates derived by reductive cleavage of S-adenosylmethionine. The primary nature of the radical intermediates makes them highly reactive and potent oxidants. They are used to initiate biotransformations by hydrogen atom abstraction, a process that allows a particularly diverse range of substrates to be functionalized, including substrates with relatively inert chemical structures. In the first part of this review, we discuss the evidence supporting the mechanism of radical formation from S-adenosylmethionine. In the second part of the review, we examine the potential of reaction products arising from S-adenosylmethionine to cause product inhibition. The effects of this product inhibition on kinetic studies of 'radical S-adenosylmethionine' enzymes are discussed and strategies to overcome these issues are reviewed. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Assuntos
Coenzimas/metabolismo , Retroalimentação Fisiológica , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coenzimas/química , Desoxiadenosinas/química , Desoxiadenosinas/metabolismo , Eucariotos , Radicais Livres/química , Radicais Livres/metabolismo , Proteínas Ferro-Enxofre/química , Cinética , Modelos Moleculares , Oxirredução , Prótons , S-Adenosilmetionina/química , TermodinâmicaRESUMO
Polysilicon nanowire biosensors have been fabricated using a top-down process and were used to determine the binding constant of two inflammatory biomarkers. A very low cost nanofabrication process was developed, based on simple and mature photolithography, thin film technology, and plasma etching, enabling an easy route to mass manufacture. Antibody-functionalized nanowire sensors were used to detect the proteins interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) over a wide range of concentrations, demonstrating excellent sensitivity and selectivity, exemplified by a detection sensitivity of 10 fM in the presence of a 100,000-fold excess of a nontarget protein. Nanowire titration curves gave antibody-antigen dissociation constants in good agreement with low-salt enzyme-linked immunosorbent assays (ELISAs). This fabrication process produces high-quality nanowires that are suitable for low-cost mass production, providing a realistic route to the realization of disposable nanoelectronic point-of-care (PoC) devices.
Assuntos
Técnicas Biossensoriais/instrumentação , Membranas Artificiais , Nanofios/química , Polímeros/química , Silício/química , Reações Antígeno-Anticorpo , Biomarcadores/análise , Cristalização , Ensaio de Imunoadsorção Enzimática , Inflamação , Interleucina-8/análise , Interleucina-8/imunologia , Polímeros/síntese química , Propriedades de Superfície , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/imunologiaRESUMO
N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmols(-1) in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.
Assuntos
DNA/metabolismo , Fluoresceína/análise , Pyrococcus horikoshii/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/análise , Sequência de Bases , DNA/química , Metilação de DNA , Fluoresceína/química , Cinética , Pyrococcus horikoshii/enzimologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Especificidade por Substrato , Temperatura , Termodinâmica , p-Dimetilaminoazobenzeno/análogos & derivados , p-Dimetilaminoazobenzeno/análise , p-Dimetilaminoazobenzeno/químicaRESUMO
In Salmonella enterica, ThiI is a bifunctional enzyme required for the synthesis of both the 4-thiouridine modification in tRNA and the thiazole moiety of thiamine. In 4-thiouridine biosynthesis, ThiI adenylates the tRNA uridine and transfers sulfur from a persulfide formed on the protein. The role of ThiI in thiazole synthesis is not yet well understood. Mutational analysis described here found that ThiI residues required for 4-thiouridine synthesis were not involved in thiazole biosynthesis. The data further showed that the C-terminal rhodanese domain of ThiI was sufficient for thiazole synthesis in vivo. Together, these data support the conclusion that sulfur mobilization in thiazole synthesis is mechanistically distinct from that in 4-thiouridine synthesis and suggest that functional annotation of ThiI in genome sequences should be readdressed. Nutritional studies described here identified an additional cysteine-dependent mechanism for sulfur mobilization to thiazole that did not require ThiI, IscS, SufS, or glutathione. The latter mechanism may provide insights into the chemistry used for sulfur mobilization to thiazole in organisms that do not utilize ThiI.
Assuntos
Proteínas de Bactérias/metabolismo , Salmonella typhimurium/metabolismo , Sulfurtransferases/metabolismo , Tiamina/biossíntese , Tiazóis/metabolismo , Proteínas de Bactérias/genética , Meios de Cultura/química , Análise Mutacional de DNA , Modelos Biológicos , Modelos Químicos , Estrutura Terciária de Proteína , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Sulfurtransferases/genética , Terminologia como AssuntoRESUMO
In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.
Assuntos
Avidina/metabolismo , Técnicas Biossensoriais , Biotinilação , Proteínas Imobilizadas/metabolismo , Sondas de Oligonucleotídeos/metabolismo , Avidina/química , Sequência de Bases , Biotina/metabolismo , Proteínas Imobilizadas/química , Cinética , Sondas de Oligonucleotídeos/genética , Ligação Proteica , TermodinâmicaRESUMO
The radical SAM superfamily of enzymes catalyzes a broad spectrum of biotransformations by employing a common obligate intermediate, the 5'-deoxyadenosyl radical (DOA). Radical formation occurs via the reductive cleavage of S-adenosylmethionine (SAM or AdoMet). The resultant highly reactive primary radical is a potent oxidant that enables the functionalization of relatively inert substrates, including unactivated C-H bonds. The reactions initiated by the DOA are breathtaking in their efficiency, elegance and in many cases, the complexity of the biotransformation achieved. This review describes the common features shared by enzymes that generate the DOA and the intriguing variations or modifications that have recently been reported. The review also highlights selected examples of the diverse biotransformations that ensue.
Assuntos
S-Adenosilmetionina/metabolismo , Animais , Humanos , Transferases Intramoleculares/metabolismo , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/químicaRESUMO
Cfr is a radical-SAM (S-adenosyl-L-methionine) enzyme that methylates the 8 position of 23S rRNA residue A2503 to confer resistance to multiple antibiotic classes acting upon the large subunit of the bacterial ribosome. Radical-SAM enzymes use an Fe-S cluster to generate the 5'-deoxyadenosyl (DOA) radical from SAM, enabling them to modify intrinsically unreactive centres such as adenosine C8. However, despite its mechanistic interest and clinical relevance, until recently Cfr remained little characterised. Accordingly we have used co-expression with the Azotobacter vinelandii isc operon, encoding genes responsible for Fe-S cluster biosynthesis, to express hexahistidine-tagged Cfr in Escherichia coli BL21Star, and purified the recombinant protein in a yield more than 20 times greater than has been previously reported. As aerobically purified, Cfr contains secondary structure, is monomeric in solution and has an absorbance spectrum suggestive of a 2Fe-2S cluster. After anaerobic purification a 4Fe-4S cluster is indicated, while on reconstitution with excess iron and sulphide a further increase in metal content suggests that an additional, most likely 4Fe-4S, cluster is formed. Acquisition of additional secondary structure under these conditions indicates that Fe-S clusters are of structural, as well as functional, importance to Cfr. In the presence of sodium dithionite reconstituted Cfr is both reducible and able to cleave SAM to 5'-deoxyadeonsine (DOA), demonstrating that the purified reconstituted enzyme has radical-SAM activity. Co-expression with isc proteins thus enables recombinant active Cfr to be obtained in yields that facilitate its future spectroscopic and structural characterisation.
Assuntos
Metiltransferases/genética , Metiltransferases/metabolismo , RNA Ribossômico/metabolismo , S-Adenosilmetionina/metabolismo , Azotobacter vinelandii/genética , Farmacorresistência Bacteriana , Escherichia coli/genética , ÓperonRESUMO
Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin. Assays utilizing (13)C-tyrosine were analyzed by FTIR to confirm the production of HbCO and to demonstrate that the CO product was synthesized from tyrosine. CO ligation is a common feature at the active sites of the [FeFe], [NiFe], and [Fe]-only hydrogenases; however, this is the first report of the enzymatic synthesis of CO in hydrogenase maturation.
Assuntos
Monóxido de Carbono/metabolismo , Hidrogenase/metabolismo , Catálise , Clostridium , Proteínas de Escherichia coli , S-Adenosilmetionina , Transativadores , Tirosina/metabolismoRESUMO
A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R(2)) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5'-CG-3' site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.