RESUMEN
Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.
Asunto(s)
Adenosina Trifosfato/química , Amidohidrolasas/química , Proteínas Bacterianas/química , Caprolactama/química , Subunidades de Proteína/química , Pseudomonas/enzimología , Adenosina Trifosfato/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Ácido Aminocaproico/química , Ácido Aminocaproico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Caprolactama/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hidrólisis , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pseudomonas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , TermodinámicaRESUMEN
6-Aminohexanoic acid (6AHA) is a vital polymer building block for Nylon 6 production and an FDA-approved orphan drug. However, its production from cyclohexane is associated with several challenges, including low conversion and yield, and severe environmental issues. We aimed at overcoming these challenges by developing a bioprocess for 6AHA synthesis. A mixed-species approach turned out to be most promising. Thereby, Pseudomonas taiwanensis VLB120 strains harbouring an upstream cascade converting cyclohexane to either Ñ-caprolactone (Ñ-CL) or 6-hydroxyhexanoic acid (6HA) were combined with Escherichia coli JM101 strains containing the corresponding downstream cascade for the further conversion to 6AHA. ε-CL was found to be a better 'shuttle molecule' than 6HA enabling higher 6AHA formation rates and yields. Mixed-species reaction performance with 4 g l-1 biomass, 10 mM cyclohexane, and an air-to-aqueous phase ratio of 23 combined with a repetitive oxygen feeding strategy led to complete substrate conversion with 86% 6AHA yield and an initial specific 6AHA formation rate of 7.7 ± 0.1 U gCDW -1 . The same cascade enabled 49% 7-aminoheptanoic acid yield from cycloheptane. This combination of rationally engineered strains allowed direct 6AHA production from cyclohexane in one pot with high conversion and yield under environmentally benign conditions.
Asunto(s)
Ácido Aminocaproico , Pseudomonas , Ácido Aminocaproico/metabolismo , Biocatálisis , Ciclohexanos , Pseudomonas/metabolismoRESUMEN
An important aspect of host-pathogen interactions is the interference of secreted proteins with the fibrinolytic system. Herein, we describe a modified ELISA method used to evaluate the interaction of a recombinant Schistosoma mansoni protein with plasminogen (PLG). Using this protocol, we demonstrated that a secreted protein, recombinant venom allergen-like protein 18 (rSmVAL18) acts as a plasminogen receptor increasing its activation into plasmin in the presence of the urokinase-type plasminogen activator (uPA). PLG binding was determined by immobilizing human PLG in the plate and incubating with the recombinant protein; competitive binding with a lysine analog demonstrated the interaction of the protein lysine residues with PLG Kringle domains. To assess the activation of S. mansoni recombinant protein-bound PLG, the amidolytic activity of generated plasmin was measured using the D-Val-Leu-Lys 4-nitroanilide dihydrochloride substrate.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas del Helminto/metabolismo , Plasminógeno/metabolismo , Schistosoma mansoni/metabolismo , Ácido Aminocaproico/metabolismo , Animales , Unión Competitiva , Fibrinolisina/metabolismo , Humanos , Unión ProteicaRESUMEN
Production of platform chemicals from renewable feedstocks is becoming increasingly important due to concerns on environmental contamination, climate change, and depletion of fossil fuels. Adipic acid (AA), 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD) are key precursors for nylon synthesis, which are currently produced primarily from petroleum-based feedstocks. In recent years, the biosynthesis of adipic acid from renewable feedstocks has been demonstrated using both bacterial and yeast cells. Here we report the biocatalytic conversion/transformation of AA to 6-ACA and HMD by carboxylic acid reductases (CARs) and transaminases (TAs), which involves two rounds (cascades) of reduction/amination reactions (AA â 6-ACA â HMD). Using purified wild type CARs and TAs supplemented with cofactor regenerating systems for ATP, NADPH, and amine donor, we established a one-pot enzyme cascade catalyzing up to 95% conversion of AA to 6-ACA. To increase the cascade activity for the transformation of 6-ACA to HMD, we determined the crystal structure of the CAR substrate-binding domain in complex with AMP and succinate and engineered three mutant CARs with enhanced activity against 6-ACA. In combination with TAs, the CAR L342E protein showed 50-75% conversion of 6-ACA to HMD. For the transformation of AA to HMD (via 6-ACA), the wild type CAR was combined with the L342E variant and two different TAs resulting in up to 30% conversion to HMD and 70% to 6-ACA. Our results highlight the suitability of CARs and TAs for several rounds of reduction/amination reactions in one-pot cascade systems and their potential for the biobased synthesis of terminal amines.
Asunto(s)
Adipatos/metabolismo , Ácido Aminocaproico/metabolismo , Biocatálisis , Diaminas/metabolismo , Oxidorreductasas/metabolismo , Transaminasas/metabolismo , Bacterias/genética , Biotransformación , Clonación Molecular , Cristalografía por Rayos X , Cinética , Oxidorreductasas/química , Conformación Proteica , Especificidad por Sustrato , Transaminasas/químicaRESUMEN
The biodegradation of the nylon-6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP-dependent hydrolytic ring opening to 6-aminohexanoate. This non-natural ω-amino acid is converted to 6-oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I pyridoxal 5'-phosphate (PLP) enzymes. To understand the structural basis of 6-aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat /KM ) with the caprolactam degradation intermediates 6-aminohexanoate and 6-oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three-dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6-aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate-binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6-aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6-aminohexanoate degradation. DATABASE: The atomic coordinates and structure factors P. jessenii 6-aminohexanoate aminotransferase have been deposited in the PDB as entries 6G4B (Eâsuccinate complex), 6G4C (Eâphosphate complex), 6G4D (EâPLP complex), 6G4E (EâPLP-6-aminohexanoate intermediate), and 6G4F (EâPMP complex).
Asunto(s)
Ácido Aminocaproico/metabolismo , Proteínas Bacterianas/metabolismo , Caprolactama/metabolismo , Pseudomonas/enzimología , Fosfato de Piridoxal/metabolismo , Transaminasas/química , Transaminasas/metabolismo , Secuencia de Aminoácidos , Ácido Aminocaproico/química , Proteínas Bacterianas/química , Caprolactama/química , Cristalografía por Rayos X , Modelos Moleculares , Filogenia , Homología de Secuencia , Especificidad por SustratoRESUMEN
Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD 1 and nylE 1 , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD1) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE1) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP+ as a cofactor. Phylogenic analysis revealed that NylD1 should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE1 should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD1/NylE1 coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.
Asunto(s)
Adipatos/metabolismo , Ácido Aminocaproico/metabolismo , Arthrobacter/enzimología , Redes y Vías Metabólicas , Nylons/metabolismo , Alanina/metabolismo , Arthrobacter/genética , Proteínas Bacterianas/metabolismo , Escherichia coli , Ácido Glutámico/metabolismo , Glicina/metabolismo , Hidrolasas/metabolismo , Ingeniería Metabólica , Fosfato de Piridoxal/metabolismo , Especificidad por Sustrato , Transaminasas/metabolismoRESUMEN
An enzymatic method for 6-oxohexanoic acid production was developed using 6-aminohexanoic acid and an ω-amino group-oxidizing enzyme (ω-AOX) from Phialemonium sp. AIU 274. 6-Oxohexanoic acid was produced from 6-aminohexanoic acid with 100% yield by incubation with 0.3 U of the ω-AOX and 20 U of catalase at 30 °C for 30 h in 0.1 M potassium phosphate buffer (pH 7.0).
Asunto(s)
Ácido Aminocaproico/metabolismo , Ascomicetos/enzimología , Biotecnología/métodos , Caproatos/metabolismo , Biocatálisis , Catalasa/metabolismo , Concentración de Iones de Hidrógeno , Oxidación-Reducción , TemperaturaRESUMEN
ε-Caprolactam and δ-valerolactam are important commodity chemicals used in the manufacture of nylons, with millions of tons produced annually. Biological production of these highly valued chemicals has been limited due to a lack of enzymes that cyclize ω-amino fatty acid precursors to corresponding lactams under ambient conditions. In this study, we demonstrated production of these chemicals using ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-02301 in Streptomyces aizunensis. This enzyme has a broad substrate spectrum and can cyclize 4-aminobutyric acid into γ-butyrolactam, 5-aminovaleric acid into δ-valerolactam and 6-aminocaproic acid into ε-caprolactam. Recombinant E. coli expressing ORF26 produced valerolactam and caprolactam when 5-aminovaleric acid and 6-aminocaproic acid were added to the culture medium. Upon coexpressing ORF26 with a metabolic pathway that produced 5-aminovaleric acid from lysine, we were able to demonstrate production of δ-valerolactam from lysine.
Asunto(s)
Coenzima A Ligasas/metabolismo , Lactamas/metabolismo , Streptomyces/enzimología , Aminoácidos Neutros/metabolismo , Ácido Aminocaproico/metabolismo , Proteínas Bacterianas/metabolismo , Lisina/metabolismo , Sistemas de Lectura Abierta/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Mycoplasma pneumoniae is a common cause of community-acquired infections of the human respiratory tract. The strongly reduced genome of the cell wall-less bacteria results in limited metabolic pathways and a small number of known virulence factors. In addition to the well-characterized adhesion apparatus and the expression of tissue-damaging substances, surface-exposed proteins with a primary function in cytosol-located processes such as glycolysis have been attracting attention in recent years. Due to interactions with host factors, it has been suggested that these bacterial proteins contribute to pathogenesis. Here, we investigated the chaperones GroEL and DnaK of M. pneumoniae as candidates for such moonlighting proteins. After successful expression in Escherichia coli and production of polyclonal antisera, the localization of both chaperones on the surface of bacteria was confirmed. Binding of recombinant GroEL and DnaK to human A549 cells, to plasminogen as well as to vitronectin, fibronectin, fibrinogen, lactoferrin and laminin was demonstrated. In the presence of both recombinant proteins and host activators, plasminogen can be activated to the protease plasmin, which is able to degrade vitronectin and fibrinogen. The results of the study extend the spectrum of surface-exposed proteins in M. pneumoniae and indicate an additional role of both chaperones in infection processes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Chaperonina 60/metabolismo , Matriz Extracelular/metabolismo , Mycoplasma pneumoniae/metabolismo , Plasminógeno/metabolismo , Células A549 , Adenosina Trifosfatasas/genética , Ácido Aminocaproico/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Técnicas de Visualización de Superficie Celular , Chaperonina 60/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Sueros Inmunes/inmunología , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/inmunología , Neumonía por Mycoplasma/metabolismo , Neumonía por Mycoplasma/microbiología , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: To assess the in vitro effects of drug sequestration in extracorporeal membrane oxygenation (ECMO) on ϵ-aminocaproic acid (EACA) concentrations. METHODS AND DESIGN: This in vitro study will determine changes in EACA concentration over time in ECMO circuits. A pediatric dose of 2,500 mg was administered to whole expired blood in the simulated pediatric ECMO circuit. Blood samples were collected at 0, 30, 60, 360 and 1440-minute intervals after initial administration equilibration from three different sites of the circuit: pre-oxygenator (PRE), post-oxygenator (POST) and PVC tubing (PVC) to determine the predominant site of drug loss. The circuit was maintained for two consecutive days with a re-dose at 24 hours to establish a comparison between unsaturated (New) and saturated (Old) oxygenator membranes. Comparisons between sample sites, sample times and New versus Old membranes were statistically analyzed by a linear mixed-effects model with significance defined as a p-value <0.05. RESULTS: There were no significant differences in EACA concentration with respect to sample site, with PRE and POST samples demonstrating respective mean differences of 0.30 mg/ml and 0.34 mg/ml as compared to PVC, resulting in non-significant p-values of 0.373 [95% CI (-0.37, 0.98)] and 0.324 [95% CI (-0.34, 1.01)], respectively. The comparison of New vs. Old ECMO circuits resulted in non-significant changes from baseline, with a mean difference of 0.50 mg/ml, 95% CI (-0.65, 1.65), p=0.315. CONCLUSION: The findings of this study did not show any significant changes in drug concentration that can be attributed to sequestration within the ECMO circuit. Mean concentrations between ECMO circuit sample sites did not differ significantly. Comparison between New and Old circuits also did not differ significantly in the change from baseline concentration over time. Sequestration within ECMO circuits appears not to be a considerable factor for EACA administration.
Asunto(s)
Ácido Aminocaproico/análisis , Antifibrinolíticos/análisis , Oxigenación por Membrana Extracorpórea/instrumentación , Ácido Aminocaproico/metabolismo , Antifibrinolíticos/metabolismo , Humanos , Oxigenadores de MembranaRESUMEN
Trypanosoma evansi is a widely-distributed haemoflagellated parasite of veterinary importance that infects a variety of mammals including horses, mules, camels, buffalos, cattle and deer. It is the causal agent of a trypanosomiasis known as Surra which produces epidemics of great economic importance in Africa, Asia and South America. The main pathology includes an enlarged spleen with hypertrophy of lymphoid follicles, congested lungs, neuronal degeneration and meningoencephalitis, where migration of the parasites from the blood to the tissues is essential. Most cells, including pathogenic cells, use diverse strategies for tissue invasion, such as the expression of surface receptors to bind plasminogen or plasmin. In this work, we show that T. evansi is able to bind plasminogen and plasmin on its surface. The analysis of this binding revealed a high affinity dissociation constant (Kd of 0.080±0.009µM) and 1×10(5) plasminogen binding sites per cell. Also a second population of receptors with a Kd of 0.255±0.070µM and 3.2×10(4) plasminogen binding sites per cell was determined. Several proteins with molecular masses between â¼18 and â¼70kDa are responsible for this binding. This parasite-plasminogen interaction may be important in the establishment of the infection in the vertebrate host, where the physiological concentration of available plasminogen is around 2µM.
Asunto(s)
Fibrinolisina/metabolismo , Plasminógeno/metabolismo , Trypanosoma/metabolismo , Tripanosomiasis/veterinaria , Ácido Aminocaproico/metabolismo , Animales , Sitios de Unión , Carbonatos/farmacología , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Caballos , Sueros Inmunes/inmunología , Microsomas/química , Microsomas/efectos de los fármacos , Plasminógeno/inmunología , Proteínas Protozoarias/análisis , Conejos , Ratas , Ratas Wistar , Receptores de Superficie Celular/metabolismo , Trypanosoma/patogenicidad , Trypanosoma/fisiología , Tripanosomiasis/parasitología , Tripanosomiasis/patología , Tubulina (Proteína)/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Interactions between polyelectrolytes and oppositely charged surfactants have been in a great interest for several decades, yet the conventional surfactants may cause a problem in medical applications. Interactivity between polysaccharide hyaluronan (HA) and amino acids Lysine, 6-Aminocaproic acid (6-AcA), and Arginine as an alternative system is reported. The interactions were investigated by means of rheology and electric conductance and the electronic structures were explored by the density functional theory (DFT). Lysine exhibits the strongest interaction of all, which was manifested, e.g. by nearly 6-time drop of the initial viscosity comparing with only 1.3-time lower value in the case of 6-AcA. Arginine interaction with HA was surprisingly weaker in terms of viscosity than that of Lysine due to a lower and delocalized charge density on its guanidine group. According to the DFT calculations, the binding of Lysine to HA was found to be more flexible, while Arginine creates more rigid structure with HA.
Asunto(s)
Ácido Aminocaproico/metabolismo , Arginina/metabolismo , Ácido Hialurónico/metabolismo , Lisina/metabolismo , Ácido Aminocaproico/química , Arginina/química , Sitios de Unión , Ácido Hialurónico/química , Lisina/química , Modelos Moleculares , ViscosidadAsunto(s)
Actinobacteria/metabolismo , Caprolactama/análogos & derivados , Caprolactama/metabolismo , Polímeros/metabolismo , Acetilcoenzima A/metabolismo , Actinobacteria/aislamiento & purificación , Adipatos/metabolismo , Ácido Aminocaproico/metabolismo , Caproatos/metabolismo , Medios de Cultivo/química , Hidrólisis , Aguas del Alcantarillado/microbiología , Ácido Succínico/metabolismoRESUMEN
Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.
Asunto(s)
Caprolactama/análogos & derivados , Ingeniería Metabólica/métodos , Polímeros/síntesis química , Adipatos/metabolismo , Ácido Aminocaproico/metabolismo , Técnicas de Cultivo Celular por Lotes , Caprolactama/síntesis química , Cromatografía Liquida , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Metaboloma , Ácidos Pimélicos/metabolismo , Proteómica , Espectrometría de Masas en Tándem , Ácidos Tricarboxílicos/metabolismoRESUMEN
Optimizing bio-production involves strain and process improvements performed as discrete steps. However, environment impacts genotype and a strain that is optimal under one set of conditions may not be under different conditions. We present a methodology to simultaneously vary genetic and process factors, so that both can be guided by design of experiments (DOE). Advances in DNA assembly and gene insulation facilitate this approach by accelerating multi-gene pathway construction and the statistical interpretation of screening data. This is applied to a 6-aminocaproic acid (6-ACA) pathway in Escherichia coli consisting of six heterologous enzymes. A 32-member fraction factorial library is designed that simultaneously perturbs expression and media composition. This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly). Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease. Therefore, if genotype and media were varied separately this would lead to a suboptimal combination. This is applied to the design of a strain and media composition that increases 6-ACA from 9 to 48 mg/l in a single optimization step. This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.
Asunto(s)
Algoritmos , Ácido Aminocaproico/metabolismo , Ingeniería Metabólica/métodos , Medios de Cultivo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Redes y Vías Metabólicas/genéticaRESUMEN
The combination of two cofactor self-sufficient biocatalytic cascade modules allowed the successful transformation of cyclohexanol into the nylon-6 monomer 6-aminohexanoic acid at the expense of only oxygen and ammonia. A hitherto unprecedented carboxylic acid capping strategy was introduced to minimize the formation of the dead-end intermediate 6-hydroxyhexanoic acid. For this purpose, the precursor ε-caprolactone was converted in aqueous medium in the presence of methanol into the corresponding methyl ester instead of the acid. Hence, it was shown for the first time that esterases--specifically horse liver esterase--can perform the selective ring-opening of ε-caprolactone with a clear preference for methanol over water as the nucleophile.
Asunto(s)
Ácido Aminocaproico/metabolismo , Ciclohexanoles/metabolismo , Esterasas/metabolismo , Ácido Aminocaproico/química , Animales , Biocatálisis , Ciclohexanoles/química , Esterasas/química , Caballos , Hígado/enzimología , Estructura MolecularRESUMEN
We synthesized N-(4-nitrophenyl)-6-aminohexanamide (AHpNA) and used it as a substrate in a kinetic study of 6-aminohexanoate-hydrolase (NylB), a nylon oligomer-hydrolyzing enzyme. NylBs derived from Arthrobacter sp. KI72 and Pseudomonas sp. NK87 hydrolyzed AHpNA as well as a 6-aminohexanoic acid dimer, a known substrate for NylB. The K(m) values of the NylB from Arthrobacter sp. KI72 and Pseudomonas sp. NK87 for AHpNA were 0.5 mM and 2.0 mM, respectively.
Asunto(s)
Amidohidrolasas/metabolismo , Pruebas de Enzimas/métodos , Nitrofenoles/metabolismo , Amidohidrolasas/análisis , Aminocaproatos , Ácido Aminocaproico/química , Ácido Aminocaproico/metabolismo , Arthrobacter/enzimología , Hidrólisis , Cinética , Nitrofenoles/síntesis química , Nitrofenoles/química , Pseudomonas/enzimología , Especificidad por SustratoRESUMEN
Transdermal permeation enhancers are compounds that temporarily increase drug flux through the skin by interacting with constituents of the stratum corneum. Transkarbam 12 (T12) is a highly active, broad-spectrum, biodegradable enhancer with low toxicity and low dermal irritation. We show here that T12 acts by a dual mechanism of action. The first part of this activity is associated with its ammonium carbamate polar head as shown by its pH-dependent effects on the permeation of two model drugs. Once this ammonium carbamate penetrates into the stratum corneum intercellular lipids, it rapidly decomposes releasing two molecules of protonated dodecyl 6-aminohexanoate (DDEAC) and carbon dioxide. This was observed by thermogravimetric analysis and infrared spectroscopy. This step of T12 action influences drug permeation through lipidic pathways, not through the aqueous pores (polar pathway) as shown by its effects on various model drugs and electrical impedance. Consequently, protonated DDEAC released in the stratum corneum is also an active enhancer. It broadens the scope of T12 action since it is also able to increase permeation of hydrophilic drugs that prefer the pore pathway. Thus, this dual effect of T12 is likely responsible for its favorable properties, which make it a good candidate for prospective clinical use.
Asunto(s)
Adyuvantes Farmacéuticos/farmacología , Carbamatos/farmacología , Absorción Cutánea/efectos de los fármacos , Adyuvantes Farmacéuticos/química , Adyuvantes Farmacéuticos/metabolismo , Administración Cutánea , Aminocaproatos , Ácido Aminocaproico/química , Ácido Aminocaproico/metabolismo , Ácido Aminocaproico/farmacología , Animales , Carbamatos/química , Carbamatos/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Impedancia Eléctrica , Epidermis/química , Hidrocortisona/administración & dosificación , Hidrocortisona/metabolismo , Concentración de Iones de Hidrógeno , Lípidos/química , Lípidos/aislamiento & purificación , Ácido Palmítico/química , Permeabilidad/efectos de los fármacos , Piel/efectos de los fármacos , Piel/metabolismo , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Espectrofotometría Infrarroja , Sus scrofa , Teofilina/administración & dosificación , Teofilina/metabolismo , TermogravimetríaRESUMEN
1. Zinc acexamate (ZAC) is ionized to zinc and epsilon-acetamidocaproic acid (AACA). Thus, the pharmacokinetics and tissue distribution of zinc and AACA after intravenous (50 mg kg(-1)) and oral (100 mg kg(-1)) administration of ZAC were evaluated in rats. Also the pharmacokinetics of AACA after intravenous (10, 20, 30, and 50 mg kg(-1)) and oral (20, 50, and 100 mg kg(-1)) administration of ZAC and the first-pass extractions of AACA at a ZAC dose of 20 mg kg(-1) were evaluated in rats. 2. After oral administration of ZAC (20 mg kg(-1)), approximately 0.408% of the oral dose was not absorbed, the F value was approximately 47.1%, and the hepatic and gastrointestinal (GI) first-pass extractions of AACA were approximately 8.50% and 46.4% of the oral dose, respectively. The incomplete F value of AACA was mainly due to the considerable GI first-pass extraction in rats. 3. Affinity of rat tissues to zinc and AACA was low-the tissue-to-plasma (T/P) ratios were less than unity. The equilibrium plasma-to-blood cells partition ratios of AACA were independent of initial blood ZAC concentrations of 1, 5, and 10 microg ml(-1)-the mean values were 0.481, 0.490, and 0.499, respectively. The bound fractions of zinc and AACA to rat plasma were 96.6% and 39.0%, respectively.
Asunto(s)
Aminocaproatos , Antiulcerosos/farmacocinética , Administración Oral , Ácido Aminocaproico/administración & dosificación , Ácido Aminocaproico/sangre , Ácido Aminocaproico/química , Ácido Aminocaproico/metabolismo , Ácido Aminocaproico/farmacocinética , Animales , Antiulcerosos/administración & dosificación , Antiulcerosos/metabolismo , Relación Dosis-Respuesta a Droga , Estructura Molecular , Ratas , Distribución TisularRESUMEN
We characterized the interactions between plasminogen and neurons and investigated the associated effects on extracellular matrix proteolysis, cell morphology, adhesion, signaling and survival. Upon binding of plasminogen to neurons, the plasmin formed by constitutively expressed tissue plasminogen activator (tPA) degrades extracellular matrix proteins, leading to retraction of the neuron monolayer that detaches from the matrix. This sequence of events required both interaction of plasminogen with carboxy-terminal lysine residues and the proteolytic activity of plasmin. Surprisingly, 24h after plasminogen addition, plasmin-detached neurons survived and remained associated in clusters maintaining focal adhesion kinase phosphorylation contrasting with other adherent cell types fully dissociated by plasmin. However, long-term incubation (72 h) with plasminogen was associated with an increased rate of apoptosis, suggesting that prolonged exposure to plasmin may cause neurotoxicity. Regulation of neuronal organization and survival by plasminogen may be of pathophysiological relevance, as plasminogen is expressed in the brain and/or extravasate during vascular accidents or inflammatory processes.