RESUMEN
Onoceroids are a rare family of triterpenes. One representative onoceroid is ambrein, which is the main component of ambergris used as a traditional medicine. We have previously identified the onoceroid synthase, BmeTC, in Bacillus megaterium and succeeded in creating ambrein synthase by introducing mutations into BmeTC. Owing to the structural similarity of ambrein to vitamin D, a molecule with diverse biological activities, we hypothesized that some of the activities of ambergris may be induced by the binding of ambrein to the vitamin D receptor (VDR). We demonstrated the VDR binding ability of ambrein. By comparing the structure-activity relationships of triterpenes with both the VDR affinity and osteoclastic differentiation-promoting activity, we observed that the activity of ambrein was not induced via the VDR. Therefore, some of the activities of ambergris, but not all, can be attributed to its VDR interaction. Additionally, six unnatural onoceroids were synthesized using the BmeTC reactions, and these compounds exhibited higher VDR affinity than that of ambrein. Enzymatic syntheses of onoceroid libraries will be valuable in creating a variety of bioactive compounds beyond ambergris.
Asunto(s)
Ámbar Gris , Triterpenos , Ámbar Gris/química , Receptores de Calcitriol , Triterpenos/farmacología , Naftoles/química , Vitamina DRESUMEN
Active vitamin D form 1α,25-dihydroxtvitamin D3 (1,25(OH)2D3) plays pivotal roles in calcium homeostasis and osteogenesis via its transcription regulation effect via binding to vitamin D receptor (VDR). Mutated VDR often causes hereditary vitamin D-dependent rickets (VDDR) type II, and patients with VDDR-II are hardly responsive to physiological doses of 1,25(OH)D3. Current therapeutic approaches, including high doses of oral calcium and supraphysiologic doses of 1,25(OH)2D3, have limited success and fail to improve the quality of life of affected patients. Thus, various vitamin D analogues have been developed as therapeutic options. In our previous study, we generated genetically modified rats with mutated Vdr(R270L), an ortholog of human VDR(R274L) isolated from the patients with VDDR-II. The significant reduced affinity toward 1,25(OH)2D3 of rat Vdr(R270L) enabled us to evaluate biological activities of exogenous VDR ligand without 1α-hydroxy group such as 25(OH)D3. In this study, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exerted much higher affinity for Vdr(R270L) in in vitro ligand binding assay than both 25(OH)D3 and 1,25(OH)2D3. A robust osteogenic activity of AH-1 was observed in Vdr(R270L) rats. Only a 40-fold lower dose of AH-1 than that of 25(OH)D3 was effective in ameliorating rickets symptoms in Vdr(R270L) rats. Therefore, AH-1 may be promising for the therapy of VDDR-II with VDR(R274L).
Asunto(s)
Receptores de Calcitriol , Raquitismo , Animales , Calcio , Humanos , Ligandos , Osteogénesis , Calidad de Vida , Ratas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Raquitismo/metabolismo , Vitamina DRESUMEN
The inside of living cells is crowded by extremely high concentrations of biomolecules, and thus globular proteins should have been developed to increase their solubility under such crowding conditions during organic evolution. The O2-storage protein myoglobin (Mb) is known to be expressed in myocytes of diving mammals in much larger quantities than those of land mammals. We have previously resurrected ancient whale and pinniped Mbs and experimentally demonstrated that the diving animal Mbs have evolved to maintain high solubility under the crowding conditions or to increase their tolerance against macromolecular precipitants, rather than solubility in a dilute buffer solution. However, the detail of chemical mechanisms of the precipitant tolerance remains unclear. Here, we investigated pH dependence of the precipitant tolerance (ß, slope of the solubility against precipitant concentration) of extant Mbs and plotted the ß values, as well as those of ancestral Mbs, against their surface net charges (ZMb). The results demonstrated that the precipitant tolerance was approximated by the square of ZMb, that is, ß = aZMb2 + b, in which a and b are constants. This effect of ZMb against the precipitation is not predicted by a classical excluded volume theory that gives constant ß for Mbs but can be explained by electrostatic repulsion between Mb molecules. The present study elucidates how Mb molecules have evolved to increase their in vivo solubility and shows the physiological significance of either neutral or basic isoelectric points (pI) of the natural Mbs, rather than acidic pI.
Asunto(s)
Mamíferos , Mioglobina , Animales , Sustancias Macromoleculares , Mamíferos/metabolismo , Mioglobina/química , Solubilidad , Electricidad EstáticaRESUMEN
Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.
Asunto(s)
Aptámeros de Nucleótidos , Neoplasias , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/química , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Aptámeros de Nucleótidos/farmacología , Colecalciferol/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Simulación del Acoplamiento Molecular , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Vitamina D3 24-Hidroxilasa/genética , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
Myoglobin (Mb) is highly concentrated in the myocytes of diving mammals such as whales and seals, in comparison with land animals, and its molecular evolution has played a crucial role in their deep-sea adaptation. We previously resurrected ancestral whale Mbs and demonstrated the evolutional strategies for higher solubility under macromolecular crowding conditions. Pinnipeds, such as seals and sea lions, are also expert diving mammals with Mb-rich muscles. In the present study, we resurrected ancestral pinniped Mbs and investigated their biochemical and structural properties. Comparisons between pinniped and whale Mbs revealed the common and distinctive strategies for the deep-sea adaptation. The overall evolution processes, gaining precipitant tolerance and improving thermodynamic stability, were commonly observed. However, the strategies for improving the folding stability differed, and the pinniped Mbs exploited the shielding of hydrophobic surfaces more effectively than the whale Mbs.
RESUMEN
Extant cetaceans, such as sperm whale, acquired the great ability to dive into the ocean depths during the evolution from their terrestrial ancestor that lived about 50 million years ago. Myoglobin (Mb) is highly concentrated in the myocytes of diving animals, in comparison with those of land animals, and is thought to play a crucial role in their adaptation as the molecular aqualung. Here, we resurrected ancestral whale Mbs, which are from the common ancestor between toothed and baleen whales (Basilosaurus), and from a further common quadrupedal ancestor between whale and hippopotamus (Pakicetus). The experimental and theoretical analyses demonstrated that whale Mb adopted two distinguished strategies to increase the protein concentration in vivo along the evolutionary history of deep sea adaptation; gaining precipitant tolerance in the early phase of the evolution, and increase of folding stability in the late phase.
Asunto(s)
Evolución Molecular , Mioglobina/genética , Ballenas/metabolismo , Secuencia de Aminoácidos , Animales , Extinción Biológica , Mioglobina/química , Oxígeno/metabolismo , Filogenia , ProbabilidadRESUMEN
Proteins are attractive materials for supramolecular chemistry due to their multifunctionality and self-organization ability. In this work, we synthesized a diheme compound, in which two iron-protoporphyrin IX molecules are associated via a linker chain, and introduced it into a de novo designed four-helix bundle protein with two heme-binding sites. The protein gradually bound the diheme compound by bis-histidyl ligation and formed supramolecular polymers. Polymer formation was observed by atomic force microscopy (AFM), which revealed the highly branched, dendritic forms of the fibrous architecture. The present results may open a pathway toward nanowire construction with de novo heme-proteins.
RESUMEN
Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract in the presence of gallic acid are thus quite different from those of nonlaccase-treated green tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are present in higher concentrations. Additionally, laccase-treated green tea extract contains further polymerized catechin derivatives, comparable with naturally fermented teas such as oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro match well with the results obtained by docking simulations of the compounds with their target enzymes or noncatalytic protein. Thus, ETFGg and laccase-treated green tea extracts containing ETFGg are promising functional food materials with potential antiobesity and antiperiodontal disease activities.
Asunto(s)
Benzocicloheptenos/química , Camellia sinensis/química , Ácido Gálico/química , Lacasa/química , Extractos Vegetales/química , Polifenoles/química , Biocatálisis , Inhibidores Enzimáticos/química , Lipasa/antagonistas & inhibidores , Lipasa/química , Oxidación-ReducciónRESUMEN
HT1 (HIGH LEAF TEMPERATURE 1) is the first component associated with changes in stomatal aperture in response to CO2 to be isolated by forward genetic screening. The HT1 gene encodes a protein kinase expressed mainly in guard cells. The loss-of-function ht1-1 and ht1-2 mutants in Arabidopsis thaliana have CO2-hypersensitive stomatal closure with concomitant reductions in their kinase activities in vitro In addition to these mutants, in this study we isolate or obtaine five new ht1 alleles (ht1-3, ht1-4, ht1-5, ht1-6, and ht1-7). Among the mutants, only ht1-3 has a dominant mutant phenotype and has widely opened stomata due to CO2 insensitivity. The ht1-3 mutant has a missense mutation affecting a non-conserved residue (R102K), whereas the other six recessive mutants have mutations in highly conserved residues in the catalytic domains required for kinase activity. We found that the dominant mutation does not affect the expression of HT1 or the ability to phosphorylate casein, a universal kinase substrate, but it does affect autophosphorylation activity in vitro A 3D structural model of HT1 also shows that the R102 residue protrudes from the surface of the kinase, implying a role for the formation of oligomers and/or interaction with its targets. We demonstrate that both the loss-of-function and gain-of-function ht1 mutants have completely disrupted CO2 responses, although they have normal responses to ABA. Furthermore, light-induced stomatal opening is smaller in ht1-3 and much smaller in ht1-2 Taken together, these results indicate that HT1 is a critical regulator for CO2 signaling and is partially involved in the light-induced stomatal opening pathway.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Dióxido de Carbono/metabolismo , Mutación , Proteínas de Plantas/genética , Proteínas Quinasas/genética , Transducción de Señal , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estomas de Plantas/enzimología , Estomas de Plantas/fisiología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Alineación de SecuenciaRESUMEN
The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dióxido de Carbono/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Sustitución de Aminoácidos , Arabidopsis/citología , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Estomas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
The antibiotic acylases belonging to the N-terminal nucleophile hydrolase superfamily are key enzymes for the industrial production of antibiotic drugs. Cephalosporin acylase (CA) and penicillin G acylase (PGA) are two of the most intensively studied enzymes that catalyze the deacylation of ß-lactam antibiotics. On the other hand, aculeacin A acylase (AAC) is known to be an alternative acylase class catalyzing the deacylation of echinocandin or cyclic lipopeptide antibiotic compounds, but its structural and enzymatic properties remain to be explored. In the present study, 3D homology models of AAC were constructed, and docking simulation with substrate ligands was performed for AAC, as well as for CA and PGA. The docking models of AAC with aculeacin A suggest that AAC has the deep narrow binding pocket for the long-chain fatty acyl group of the echinocandin molecule. To confirm this, CA mutants have been designed to form the binding pocket for the long acyl chain. Experimentally synthesized mutant enzymes exhibited lower enzymatic activity for cephalosporin but higher activity for aculeacin A, in comparison with the wild-type enzyme. The present results have clarified the difference in mechanisms of substrate selection between the ß-lactam and echinocandin acylases and demonstrate the usefulness of the computational approaches for engineering the enzymatic properties of antibiotic acylases.
Asunto(s)
Actinobacteria/enzimología , Amidohidrolasas/metabolismo , Antibacterianos/metabolismo , Equinocandinas/metabolismo , Penicilina Amidasa/metabolismo , Pseudomonas/enzimología , beta-Lactamas/metabolismo , Actinobacteria/química , Acilación , Amidohidrolasas/química , Secuencia de Aminoácidos , Sitios de Unión , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Penicilina Amidasa/química , Pseudomonas/química , Especificidad por SustratoRESUMEN
We generated and characterized giant spheroplasts from the aerobic anoxygenic photosynthetic marine bacterium Roseobacter litoralis. The giant spheroplasts contained vacuole-like structures within the cells, mainly consisting of a single membrane. The in vivo absorption spectrum of the giant spheroplasts did not have peaks typically observed for bacteriochlorophyll a. The culture media pH decreased during the growth of the giant spheroplasts. The change in the pH profile for cells grown under light was no different from that for cells grown in the dark. These results showed that the R. litoralis giant spheroplasts formed lost their photosynthetic apparatus in culture. Most of the giant spheroplasts returned to their original size, likely via filamentous cells. The culture media pH increased during the growth of the filamentous cells. Some filamentous cells had septum-like structures. In such filamentous cells, DNA was separated. Initially, the color of the separated cells was white. Two weeks later, the cells changed to red in the dark, and the in vivo absorption spectrum of the cells had peaks typically observed for bacteriochlorophyll a. Our findings strongly suggest that the giant spheroplasts of R. litoralis can control the genetic information, return to their original cell size, and regain their original functions.
RESUMEN
To extract protein-protein interaction from experimental small-angle scattering of proteins in solutions using liquid state theory, a model potential consisting of a hard-sphere repulsive potential and the excess interaction potential has been introduced. In the present study, we propose a model-potential-free integral equation method that extracts the excess interaction potential by using the experimental small-angle scattering data without specific model potential such as the Derjaguin-Landau-Verwey-Overbeek (DLVO)-type model. Our analysis of experimental small-angle X-ray scattering data for lysozyme solution shows both the stabilization of contact configurations of protein molecules and a large activation barrier against the formation of the contact configurations in addition to the screened Coulomb repulsion. These characteristic features, which are not well-described by the DLVO-type model, are interpreted as solvent effects.
Asunto(s)
Modelos Moleculares , Muramidasa/química , Muramidasa/metabolismo , Dispersión del Ángulo Pequeño , Solventes/farmacología , Unión Proteica/efectos de los fármacos , Soluciones , Difracción de Rayos XRESUMEN
Toward understanding intermolecular interactions governing self-association of proteins, the present study investigated a model protein, myoglobin, using a small-angle X-ray scattering technique. It has been known that removal of the heme makes myoglobin aggregation-prone. The interparticle interferences of the holomyoglobin and the apomyoglobin were compared in terms of the structure factor. Analysis of the structure factor using a model potential of Derjaguin-Laudau-Verwey-Overbeek (DLVO) suggests that the intermolecular interaction potential of apomyoglobin is more attractive than that of holomyoglobin at short range from the protein molecule.
Asunto(s)
Hemo/aislamiento & purificación , Mioglobina/química , Mioglobina/aislamiento & purificación , Dispersión de RadiaciónRESUMEN
It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + ß proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and ß-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the ß-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain ß-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the ß-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices.
Asunto(s)
Bacteriófago lambda/química , Proteínas Represoras/química , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Dicroismo Circular , Escherichia coli K12/química , Escherichia coli K12/genética , Escherichia coli K12/virología , Datos de Secuencia Molecular , Desnaturalización Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Desplegamiento Proteico , Espectroscopía Infrarroja por Transformada de Fourier/métodos , TermodinámicaRESUMEN
We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K(m) and V(max) values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium.
Asunto(s)
2-Propanol , Alcohol Deshidrogenasa/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Alcoholes/metabolismo , Medios de Cultivo/química , Ingeniería Genética/métodos , 2-Propanol/análisis , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Alcoholes/química , Biocatálisis , Biotecnología/métodos , Escherichia coli/enzimología , Escherichia coli/genética , Hidrógeno/metabolismo , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Agua/análisisRESUMEN
Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.
Asunto(s)
Amidohidrolasas/biosíntesis , Fermentación , Péptidos Cíclicos/metabolismo , Streptomyces/enzimología , Amidohidrolasas/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Medios de Cultivo/química , Equinocandinas/química , Equinocandinas/metabolismo , Lipopéptidos/química , Lipopéptidos/metabolismo , Micafungina , Péptidos Cíclicos/química , Streptomyces/clasificaciónRESUMEN
FR901379 acylase, an enzyme that catalyzes the hydrolysis of the palmitoyl moiety of the antifungal lipopeptide FR901379, was purified from the culture broth of Streptomyces sp. no. 6907 (FERM BP-5809), revealing the 80 kDa, two-subunit heterodimeric protein characteristic of the ß-lactam acylase family. Using oligodeoxyribonucleotide primers constructed on the basis of the N-terminal amino acid sequence of each purified subunit, the gene was identified from a cosmid library of Streptomyces sp. no. 6907 DNA. The deduced 775 amino acid sequence corresponded to a single polypeptide chain containing two subunits, and it shared 41.7% identity with aculeacin A acylase from Actinoplanes utahensis NRRL12052. FR901379 acylase activity was found to be 250-fold higher in the recombinant Streptomyces lividans 1326 carrying the cloned gene than in the original Streptomyces sp. no. 6907 strain.
Asunto(s)
Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Clonación Molecular , Péptidos Cíclicos/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Amidohidrolasas/química , Amidohidrolasas/aislamiento & purificación , Cartilla de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa , Subunidades de Proteína , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
It has recently been demonstrated that pressure induces folding of the alpha-helix of an alanine-based peptide (AK20), which is a monomer in water (Imamura and Kato, Proteins 2009;76:911-918). The present study focused on a coiled coil peptide GCN4-p1, the alpha-helices of which associate via a hydrophobic core, to examine whether the pressure stability of the alpha-helices depends on the hydrophobic core. Fourier transform infrared spectroscopy was used to investigate the effect of pressure on the secondary structures of GCN4-p1. The infrared spectra of GCN4-p1 shows the two amide I' peaks at approximately 1650 and approximately 1630 cm(-1) stemming from the solvent-inaccessible alpha-helix and the solvent-accessible alpha-helix, respectively. The intensities of both the peaks increase with increasing pressure, whereas they decrease with increasing temperature. This indicates that pressure induces both the alpha-helices of GCN4-p1 to fold. The present result suggests that the positive volume change upon unfolding of an alpha-helix is a common characteristic of peptides. The pressure-induced stabilization of the alpha-helices is discussed in comparison with the pressure denaturation of proteins.