RESUMO
Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem Fe(B) centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.
Assuntos
Oxirredutases/química , Oxirredutases/síntese química , Animais , Cristalização , Ferro/metabolismo , Modelos Moleculares , Mioglobina/química , Óxido Nítrico/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis.
Assuntos
Oxirredutases/síntese química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/síntese química , Biologia Sintética/métodos , Oxirredução , Oxirredutases/química , Proteínas Recombinantes/químicaRESUMO
Cinnamaldehyde derivatives were synthesized in good to excellent yields in one step by a mild and selective, base-free palladium(II)-catalyzed oxidative Heck reaction starting from acrolein and various arylboronic acids. Prepared α,ß-unsaturated aldehydes were used for synthesis of novel α-aryl substituted fosmidomycin analogues, which were evaluated for their inhibition of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase. IC(50) values between 0.8 and 27.3 µM were measured. The best compound showed activity comparable to that of the most potent previously reported α-aryl substituted fosmidomycin-class inhibitor.
Assuntos
Acroleína/análogos & derivados , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/síntese química , Antituberculosos/química , Antituberculosos/síntese química , Fosfomicina/análogos & derivados , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/síntese química , Mycobacterium tuberculosis/química , Oxirredutases/antagonistas & inibidores , Oxirredutases/síntese química , Acroleína/síntese química , Acroleína/química , Antituberculosos/farmacologia , Catálise , Fosfomicina/síntese química , Fosfomicina/química , Fosfomicina/farmacologia , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Oxirredução , Paládio/química , Ligação ProteicaRESUMO
Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well as chemzymes that catalyze conjugate additions, cycloadditions, and self-replicating processes. The focus will be mainly on cyclodextrin-based chemzymes since they have shown to be good candidate structures to base an enzyme model skeleton on. In addition hereto, other molecules that encompass binding properties will also be presented.
Assuntos
Esterases/síntese química , Glicosídeo Hidrolases/síntese química , Oxirredutases/síntese química , Sítios de Ligação , Catálise , Ciclodextrinas/síntese química , Ciclodextrinas/química , Esterases/química , Glicosídeo Hidrolases/química , Cinética , Modelos Químicos , Oxirredutases/químicaRESUMO
Porphyrins 1ab and 2ab were successfully synthesized from cis-alpha2-bisimidazole-beta-imidazole-tail porphyrins and two newly synthesized imidazole pickets containing an aliphatic ester chain following a [2+1] approach. The four compounds possess a distal trisimidazole set, a distal carboxylic acid, and a proximal imidazole, which constitute all the key features of the coordination environment of the active site in Bacterial Nitric Oxide Reductase (NOR) and make them the closest synthetic NOR model ligands to date.
Assuntos
Modelos Moleculares , Oxirredutases/síntese química , Sítios de Ligação , Oxirredutases/químicaRESUMO
A series of fluconazole (1) analogues, compounds 3a-k, were prepared as potential antifungal agents. They were designed by computational docking experiments to the active site of the cytochrome P450 14alpha-sterol demethylase (CYP51), whose crystal structure is known. Preliminary biological tests showed that most of the target compounds exhibit significant activities against the eight most-common pathogenic fungi. Thereby, the most potent congener, 1-[(4-tert-butylbenzyl)(cyclopropyl)amino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (3j), was found to exhibit a broad antifungal spectrum, being more active against Candida albicans, Candida tropicalis, Cryptococcus neoformans, Microsporum canis, and Trichophyton rubrum (MIC80 < 0.125 microg/ml) than the standard clinical drug itraconazole (2). The observed affinities of the lead molecules towards CYP51 indicate that a cyclopropyl residue enhances binding to the target enzyme. Our results may provide some guidance for the development of novel triazole-based antifungal lead structures.
Assuntos
Antifúngicos/síntese química , Sistema Enzimático do Citocromo P-450/síntese química , Desenho de Fármacos , Oxirredutases/síntese química , Triazóis/síntese química , Antifúngicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Química Farmacêutica/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Hidrogênio , Testes de Sensibilidade Microbiana/métodos , Oxirredutases/metabolismo , Esterol 14-Desmetilase , Triazóis/farmacologiaRESUMO
NAD(P)-dependent dehydrogenases are useful catalysts for the synthesis of chiral compounds. Many active and stable enzymes are available that (with high enantioselectivity) reduce ketones or keto acids to chiral alcohols, hydroxy acids or amino acids. For economic reasons, these reactions need coupling to the simultaneous regeneration of NAD(P)H. For preparative applications, three components have to be combined: an appropriate enzyme, an efficient coenzyme-regenerating step and a suitable reaction-engineering technique.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/síntese química , Oxirredutases/química , Álcoois/metabolismo , Leucina/metabolismo , Modelos Químicos , Oxirredução , Oxirredutases/síntese químicaRESUMO
Advances in organic chemistry now make it possible for computer-controlled instruments to synthesize new and unnatural genes. When these genes are inserted into bacterial plasmids they can code for the over-production of new, synthetic enzyme proteins. One use for these new enzymes is as catalysts for the production of only a single one of the possible optical isomers of unnatural chemicals--a technology required for the synthesis of purer and safer drugs. The article describes attempts to use this new technology to design a new enzyme which recognizes a -CH2 -COO- side chain instead of the -CH3 side chain recognized by the naturally evolved enzyme. One of the new designs is shown to enable the construction of a new enzyme which is at least as catalytically active as the natural enzyme, and in which the substrate specificity has been shifted 10(7)-fold in favour of the new target molecule. Thus, given a firm understanding of the structural properties of a natural enzyme, which determine its efficiency as a catalyst, it can be manipulated to accept a new substrate and it is reasonably hopeful that useful products will be cheaply available from the redesign of natural proteins.
Assuntos
Oxirredutases/síntese química , Engenharia Biomédica , Especificidade por SubstratoRESUMO
Three methods of synthesis of immunoglobulin conjugates with malate, lactate and glucose-6-phosphate dehydrogenases, involving the sodium metaperiodate oxidation of immunoglobulin carbohydrate component, use of water-soluble carbodiimide and the one-step glutaraldehyde technique, were compared. The glutaraldehyde method was shown to give immunoglobulin-dehydrogenase conjugates with high catalytic and immunochemical activity, which may be useful for enzyme-immunoassay.
Assuntos
Técnicas Imunoenzimáticas , Imunoglobulinas/síntese química , Oxirredutases/síntese química , Fenômenos Químicos , QuímicaAssuntos
Oxirredutases/síntese química , Oxirredutases do Álcool/análise , Animais , Autoanálise/instrumentação , Bovinos , Etanol/análise , L-Lactato Desidrogenase/análise , Malato Desidrogenase/análise , Miocárdio/enzimologia , Oxaloacetatos/análise , Oxirredutases/análise , Piruvatos/análise , Espectrofotometria , LevedurasRESUMO
We report the synthesis and structural and magnetic characterization of an original Cu(3) oxidase model. The Schiff base ligand used in the synthesis derives from condensation of acetylacetone with glycine amino acid. The K[Cu(3)(L)(3)(micro(3)-OH)].(H(2)O)(2) complex crystallizes at room temperature in the tetragonal P43212 space group with a = 20.540(3) A and c = 15.866(6) A and consists of triangular Cu(3) units. The magnetic behavior interpretation suggests the presence of spin frustration, which has been investigated by means of ab initio DDCI calculations. It is shown that the system should be viewed as a "ménage à trois" spin-coupled pattern mediated by a central hydroxo group, lifting the doublet degeneracy by approximately 8 cm-1.
Assuntos
Ferro/química , Magnetismo , Modelos Moleculares , Oxirredutases/síntese química , Cristalografia por Raios X , Oxirredutases/química , Conformação ProteicaRESUMO
The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.
Assuntos
Inositol/química , Oxirredutases/química , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sítios de Ligação , Configuração de Carboidratos , Catálise , Inositol/síntese química , Inositol/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Oxirredutases/síntese química , Oxirredutases/genética , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The semisynthetic enzyme 6 was prepared by alkylation of the cysteine-25 sulfhydryl group of papain with the bipyridine 5 and was shown to stoichiometrically bind copper ion; 7 catalyzed the autoxidation of ascorbic acid derivatives with saturation kinetics approximately 20-fold faster than a model system using 3-Cu(II).
Assuntos
Ácido Ascórbico/metabolismo , Oxirredutases/metabolismo , Cinética , Oxirredução , Oxirredutases/síntese químicaRESUMO
Herein we demonstrate the successful photochemical generation of nitric oxide via two-photon excitation (TPE) from the supramolecular complex PPIX-RSE ({mu-S,mu-S'-protoporphyrin-IX-bis(2-thioethyl)diester]tetranitrosyl-diiron). The TPE fluorescence spectra indicate efficient energy transfer from the PPIX antennae to the iron sulfur nitrosyl cluster. Further evidence of NO release is demonstrated using a nitric oxide specific electrode and ESI+ MS.
Assuntos
Oxirredutases/síntese química , Eletrodos , Raios Infravermelhos , Oxirredutases/química , Fotoquímica , Fótons , Fármacos Fotossensibilizantes/química , Protoporfirinas/química , Espectrometria de FluorescênciaRESUMO
1-Deoxy-D-xylulose-5-phosphate (DXP) is a key intermediate in the non-mevalonate pathway to terpenoids in bacteria, and it is the substrate for the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP-R). In order to study the mechanism of DXP-R, we required a flexible synthesis of the substrate which would allow the incorporation of isotopic labels, and the variation of the two stereocentres. Thus 1,4-dihydroxypent-2-yne was selectively reduced to give the E-olefin, and selective phosphorylation of the primary alcohol followed by oxidation of the secondary alcohol gave a substrate suitable for dihydroxylation. Dihydroxylation using stoichiometric OsO4 in the presence of chiral ligands gave protected DXP in high ee. Final hydrogenolysis gave DXP in quantitative yield and high purity. DXP-R was produced by rapid cloning of the dxr gene from Escherichia coli through controlled expression and ion exchange chromatography. The synthetic DXP was fully active in enzyme assays catalysed by recombinant DXP-R.
Assuntos
Aldose-Cetose Isomerases/síntese química , Complexos Multienzimáticos/síntese química , Oxirredutases/síntese química , Pentosefosfatos/síntese química , Álcoois/química , Aldose-Cetose Isomerases/química , Catálise , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Modelos Químicos , Complexos Multienzimáticos/química , Oxirredutases/química , Pentosefosfatos/química , Fosforilação , Fatores de TempoRESUMO
Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis.
Assuntos
Enzimas/síntese química , L-Lactato Desidrogenase/química , Malato Desidrogenase/síntese química , Oxirredutases/síntese química , Sequência de Aminoácidos , Sítios de Ligação , Desenho de Fármacos , Enzimas/química , Ligação de Hidrogênio , Hidroxiácidos/metabolismo , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/genética , Conformação ProteicaRESUMO
Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.
Assuntos
Hemeproteínas/química , Histidina/química , Oxirredutases/química , Tirosina/química , Sítios de Ligação , Monóxido de Carbono/química , Grupo dos Citocromos b , Citocromos/química , Dimerização , Proteínas de Escherichia coli/síntese química , Proteínas de Escherichia coli/química , Radicais Livres/química , Hemeproteínas/síntese química , Modelos Químicos , Modelos Moleculares , Oxirredutases/síntese química , Espectrofotometria Infravermelho/métodosRESUMO
Apoascorbate oxidase has been shown to have a molecular weight of 137,000 +/- 3,000 and essentially the same gross quaternary conformation as native ascorbate oxidase. The apoenzyme, however, lacks much of the conformational stability of the native enzyme. The removal of the copper from the oxidase protein, and the simultaneous reduction of the disulfide bonds results in an apoenzyme of lower structural stability than the native oxidase. The aging of apoascorbate oxidase has been found to involve a loss of ionizable tyrosine residues and a dissociation to subunits and component polypeptide chains, which was not observed with the more stable native and holo enzymes. The molecular weight of holoascorbate oxidase has been determined to be 285,000. An s020, w of 9.79 has been determined for the holoenzyme. Holoascorbate oxidase has been shown to have an electrophoretic mobility on polyacrylamide gels that is 23% lower than either the native or apoenzyme. Furthermore, electrophoresis of the holoenzyme, in buffers containing dodecyl sulfate, and also isoelectric focusing of the holenzyme, produce patterns of greater similarity to those of apoascorbate oxidase than the native enzyme.
Assuntos
Ascorbato Oxidase , Oxirredutases , Apoenzimas/análise , Apoenzimas/síntese química , Ascorbato Oxidase/síntese química , Ascorbato Oxidase/isolamento & purificação , Ascorbato Oxidase/metabolismo , Centrifugação com Gradiente de Concentração , Fenômenos Químicos , Química , Cromatografia em Gel , Cobre/análise , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Peso Molecular , Oxirredutases/síntese química , Plantas/enzimologia , Proteínas/análise , Dodecilsulfato de Sódio , Triptofano/análise , Tirosina/análiseRESUMO
A series of arylpiperazine derivatives were prepared and evaluated for their alpha1-adrenoceptor antagonistic activities and 5alpha-reductase inhibitory activities. SAR study led to the identification of the potent dual-acting compound 2f, which had a pA2 value of 7.5 for alpha1-adrenoceptor antagonism and an IC50 value of 1.5 nM for 5alpha-reductase inhibition.