RESUMO
The crystal structures of porcine pancreatic alpha-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 angstroms, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface. V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder. This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments. A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.
Assuntos
Inibidores Enzimáticos/química , Peptídeos/química , Streptomyces/metabolismo , Trissacarídeos/química , alfa-Amilases/antagonistas & inibidores , Animais , Configuração de Carboidratos , Metabolismo dos Carboidratos , Sequência de Carboidratos , Cristalização , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos , Suínos , Trissacarídeos/isolamento & purificação , Trissacarídeos/metabolismo , alfa-Amilases/químicaRESUMO
The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.
Assuntos
Peptídeos , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Modelos Moleculares , Conformação Proteica , Difração de Raios XRESUMO
Complete sequence-specific assignments of the 1H-NMR spectrum of a fusion protein of the alpha-amylase inhibitor tendamistat from Streptomyces tendae and the activation domain of Tat from human immunodeficiency virus type 1 (HIV-1) was obtained by homonuclear two-dimensional NMR methods. The protein behaves as expected for an ideal fusion protein: the flexible linker allows an almost completely decoupled motion of the subunits of the protein and the two subunits show almost no mutual interaction. In the tendamistat part, small structural distortions due to exchange of the carboxy-terminal leucine propagate mainly via the hydrogen bonds of the beta-sheet and the disulfide bond. The Tat part of the protein contains the seven cysteine residues of full-length Tat. The fusion protein was expressed in Streptomyces lividans and exported. During the export to the extracellular space disulfide bonds are created by the expressing cells, only one sulfhydryl group remains accessible for sulfhydryl reagents. Although a unique, dominant conformation with a specific disulfide bonding pattern exists, a significant conformational variation can be observed including cis-proline peptide bonds, which may indicate smaller populations with alternative disulfide bonding patterns.
Assuntos
Produtos do Gene tat/química , HIV-1/química , Peptídeos/química , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Escherichia coli , Produtos do Gene tat/genética , HIV-1/genética , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Streptomyces/química , Enxofre/química , Temperatura , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
A novel polypeptide inhibitor, AI-3688, which acts upon human pancreatic alpha-amylase, was isolated from fermentation broth of Streptomyces aureofaciens. The purified peptide contains no unusual amino acids. Its Mr is 3936. The primary structure of AI-3688 was elucidated by automatic Edman degradation of the native or modified inhibitor. Two intramolecular cysteines form a disulphide bridge, thus creating a ring structure consisting of 17 amino acids. Strong sequence homology also exists to another microbial alpha-amylase inhibitor, tendamistat (HOE 467). This paper discusses the role of a common partial sequence, -Gln-Ser-Trp-Arg-Tyr-, present in the loop of both inhibitors as the active site of microbial peptide alpha-amylase inhibitors.
Assuntos
Peptídeos/isolamento & purificação , Streptomyces aureofaciens/metabolismo , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Aminoácidos/análise , Amilases/antagonistas & inibidores , Bacillus subtilis/enzimologia , Fenômenos Químicos , Química , Dissulfetos , Peptídeos e Proteínas de Sinalização Intercelular , Pâncreas/enzimologia , Peptídeos/farmacologia , Saliva/enzimologia , Ureia/farmacologiaRESUMO
The characterization of the structure of mumbaistatin (1), an effective inhibitor of the glucose-6-phosphatase system (EC 3.1.3.9), is reported. Isolation of mumbaistatin from cultures of Streptomyces sp. DSM 11641 was achieved by anion-exchange and reversed-phase chromatography. The acid-labile inhibitor was methylated for the structure determination. Single-crystal X-ray structure analysis of a triply methylated dehydration product, C31H24O11, revealed the structure of an aromatic dispirodiketal (2), a compound containing a previously undescribed ring system. Extensive 2D-NMR experiments with mumbaistatin and with the methylation products showed that mumbaistatin itself possesses the hydroxydiketodicarboxylic acid structure 1, C28H20O12, which, in the presence of acid or upon activation through methyl ester formation, undergoes self-condensation with loss of water to the dispirodiketal form (2). Mumbaistatin is an anthraquinone derivative, whose open-chain diketo form acts as a specific and powerful inhibitor of glucose-6-phosphate translocase: IC50=5 nM. The activity towards the same enzyme of the cyclized dispirodiketal derivatives is roughly one thousand times lower.
Assuntos
Antraquinonas/química , Inibidores Enzimáticos/química , Fosfotransferases/antagonistas & inibidores , Streptomyces/metabolismo , Antraquinonas/síntese química , Antraquinonas/isolamento & purificação , Antiporters , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Ciclização , Inibidores Enzimáticos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Metilação , Conformação Molecular , Proteínas de Transporte de Monossacarídeos , Espectrofotometria Ultravioleta , Streptomyces/químicaRESUMO
The structure of a new antifungal antibiotic, mulundocandin, C48H77N7O16, was elucidated by high field NMR experiments e.g., homo- and heteronuclear correlation spectra, distortionless enhancement by polarization transfer (DEPT) spectra as well as nuclear Overhauser effect. The compound is a lipopeptide antibiotic belonging to the echinocandin class.
Assuntos
Antifúngicos/isolamento & purificação , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Equinocandinas , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos/isolamento & purificaçãoRESUMO
The terpene peptide memnopeptide A (1), C76H108N16O18S, MW 1564, was isolated from a culture of the fungus Memnoniella echinata FH 2272 on casein peptone. The structure of the novel compound was elucidated with the aid of 2D NMR experiments and from amino acid analysis and mass spectrometric sequencing of the peptide. The compound consists of a known phenylspirodrimane subunit linked to the decapeptide Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro. This proline-rich peptide is a subsequence of beta-casein. From the observed absence in the literature of any other highly significant sequence homologues, memnopeptide A can be assumed to arise from metabolic products of the fungus with direct incorporation of constituents of the nutrient medium. The formation of memnopeptide A suggests this may be a mechanism for storage of amines by the fungus. Memnopeptide A has weak antibacterial activity against gram-positive bacteria and effects half-maximal activation of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2) at a concentration of 12.5 microM.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Ativadores de Enzimas/farmacologia , Fungos Mitospóricos/metabolismo , Oligopeptídeos/farmacologia , Terpenos/farmacologia , Antiporters , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fermentação , Bactérias Gram-Positivas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Fungos Mitospóricos/química , Proteínas de Transporte de Monossacarídeos , Fosfotransferases/antagonistas & inibidores , Conformação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
Four novel lipopeptide antibiotics, friulimicins A, B, C, and D, were isolated from cultures of Actinoplanes friuliensis HAG 010964 after fermentation in different nutrient media. The new compounds were separated by ion-exchange chromatography from the acidic lipopeptides of the amphomycin type also present in the culture fluid, compounds A-1437 A, B, E, and G. The principal constituent friulimicin B, C59H94N14O19, was structurally characterized by mass spectrometric investigations of its hydrolysis and partial degradation products and by sequencing of the cyclic acyl peptide. The NMR data of friulimycin B and the amphomycin constituent A-1437 B were completely assigned by a variety of 2-D experiments, and confirmed the structures determined by mass spectrometry. All 8 lipopeptides possess an identical peptide macrocycle as their central element, linked via a diaminobutyric acid N-terminal either to an acylated asparagine residue or, in the case of the amphomycin series, to an acylated aspartic acid residue. The structures of the amphomycins have now been revised to take account of the peptide framework described herein and the determined cis-configuration of the exocyclic double bond. As a consequence of their higher isoelectric points, the new compounds friulimicin A, B, C, and D have different properties than the amphomycins.
Assuntos
Actinomycetales/metabolismo , Antibacterianos/química , Peptídeos , Inibidores da Síntese de Proteínas/química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Peptidoglicano/biossíntese , Peptidoglicano/efeitos dos fármacos , Inibidores da Síntese de Proteínas/isolamento & purificaçãoRESUMO
The novel peptide feglymycin has been isolated from cultures of Streptomyces sp. DSM 11171 by solid phase extraction, size exclusion chromatography and repeated reversed-phase chromatography. The molecular weight was found to be 1900.90 g/mol and the molecular formula is C95H97Nl3O30. Feglymycin contains 13 amino acids of which four are 3-hydroxyphenylglycine and five are 3,5-dihydroxyphenylglycine residues. The structure of the linear peptide has been determined by 1H and 13C NMR spectroscopy. The sequence was confirmed by the observed mass spectroscopic fragmentation pattern. As well as having weak antibacterial activity, feglymycin inhibits the replication of the human immunodeficiency virus (HIV) in vitro.
Assuntos
Antivirais/isolamento & purificação , HIV/efeitos dos fármacos , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Streptomyces/química , Replicação Viral/efeitos dos fármacos , Antivirais/química , Antivirais/farmacologia , Cromatografia em Gel , Escherichia coli/efeitos dos fármacos , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Células Gigantes/efeitos dos fármacos , HIV/fisiologia , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Peso Molecular , Peptídeos/química , Peptídeos/farmacologia , Proteínas/química , Proteínas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacos , Streptomyces/metabolismoRESUMO
Two novel compounds, kodaistatin A, C35H34O11, molecular weight 630, and kodaistatin C, C35H34O12, molecular weight 646, have been isolated from cultures of Aspergillus terreus Thom DSM 11247 by solid-phase extraction, size-exclusion chromatography, and various preparative HPLC steps. The use of a range of 2D NMR measurements, in particular 13C-13C correlation measurements, has led to the clarification of the structure of kodaistatin A. Kodaistatin C is a hydroxylated derivative of kodaistatin A. Both natural products contain hydroxylated aspulvinones and identical highly substituted polyketide units. An X-ray single crystal structure analysis of aspulvinon E demonstrated the z-configuration at the central double bond. The kodaistatins are effective inhibitors of the glucose-6-phosphate translocase component of the glucose-6-phosphatase system (EC 3.1.3.9), an enzyme system which is important for the control of blood glucose levels. The IC50 is 80 nM for kodaistatin A and 130 nM for kodaistatin C.
Assuntos
Aspergillus/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfotransferases/antagonistas & inibidores , Animais , Antiporters , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Inibidores Enzimáticos/isolamento & purificação , Concentração Inibidora 50 , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Proteínas de Transporte de Monossacarídeos , RatosRESUMO
Two groups of new peptaibol-type antibiotics termed cephaibols have been isolated from the fungus Acremonium tubakii, DSM 12774. These 16- or 17-unit straight-chain peptides, whose structures were characterized by amino acid analyses, 2-D NMR experiments, and by mass spectrometric sequencing, have a high content of the unusual amino acids aminoisobutyric acid and isovaline. The principal constituent of the novel peptaibol mixture is cephaibol A, which is formed in abundance in cultures of the wild strain. The striking biological property of cephaibol A is its pronounced anthelmintic action and activity against ectoparasites.
Assuntos
Acremonium/química , Anti-Helmínticos/química , Anti-Helmínticos/isolamento & purificação , Antibacterianos/química , Antibacterianos/isolamento & purificação , Peptídeos , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Anti-Helmínticos/farmacologia , Antibacterianos/farmacologia , Ascaridia/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Fenômenos Químicos , Físico-Química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura MolecularRESUMO
The new pluramycin-type antibiotics pluraflavin A, C43H54N2O14, pluraflavin B, C43H56N2O15, and pluraflavin E, C36H41NO14 were isolated from cultures of the Saccharothrix species DSM 12931. The structures of the novel compounds were elucidated with the aid of 2D NMR and mass spectrometric investigations. The characteristic structural element of pluraflavins A and B is an additional 4-epi-vancosamine unit at position 13 of the anthraquinone-gamma-pyrone ring system. Pluraflavin E has a carboxyl group in this position. Pluraflavin A has a reactive dimethyl epoxide side chain at position 2 of the anthraquinone-gamma-pyrone aglycon, which may explain the high activity of the antibiotic. The outstanding biological characteristic of pluraflavin A is its powerful, organ-dependent cytostatic action: the IC50 in the colon carcinoma proliferation assay is in the subnanomolar range.
Assuntos
Actinomycetales/metabolismo , Antraquinonas/isolamento & purificação , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Antibióticos Antineoplásicos/biossíntese , Antibióticos Antineoplásicos/isolamento & purificação , Divisão Celular/efeitos dos fármacos , Actinomycetales/crescimento & desenvolvimento , Antraquinonas/química , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Meios de Cultura , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Estrutura Molecular , Células Tumorais CultivadasRESUMO
The actagardine-producing strain Actinoplanes liguriae ATCC 31048, forms an additional lantibiotic when it is cultured on mannitol and soya meal. The new compound, Ala(0)-actagardine (1), has been isolated by solid-phase extraction followed by a two-step chromatographic separation. The molecular formula of 1 is C84H129N21O25S4. Its chemical structure was determined by 2D-NMR analysis and was further confirmed by an amino acid analysis, Edman degradation, and partial synthesis from actagardine. 1 exhibits a slightly higher biological activity than the parent compound actagardine. The synthetic analogs Lys(0)-actagardine (2) and Ile(0)-actagardine (3) demonstrate also antibacterial activities and emphasize the importance of the N-terminus for further derivatization.
Assuntos
Actinomycetales/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos , Sequência de Aminoácidos , Antibacterianos/isolamento & purificação , Bacteriocinas , Fermentação , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
New antifungal antibiotics, designated as 3874 H1 and H3, were discovered in the fermentation broth of the strain Streptomyces sp. HAG 003874. The compounds were obtained as yellow powders after sequential purification by chromatography on MCI Gel CHP20P, Fractogel HW-40 and ODS reversed phase chromatography. On the basis of the results of spectroscopic analysis, it was found that 3874 H1, C58H86N2O18, MW 1098, belongs to the p-aminoacetophenone containing family of heptaene antibiotics, while 3874 H3, C57H87NO18, MW 1073, is a non-aromatic heptaene. In addition to these, a minor component, 3874 H2, C59H88N2O18, MW 1112, a N-methyl derivative of 3874 H1 has been detected. The structures were elucidated through mass spectral analyses and 1-D and 2-D homonuclear and heteronuclear NMR data. The outstanding physico-chemical feature of 3874 H3 is its improved solubility. The new heptaenes are potent antifungal compounds with broad activity spectra, encompassing dermatophytes, yeasts and filamentous fungi.