RESUMEN
Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 µM were obtained for 1a-c, respectively.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Lisina/análogos & derivados , Valina/análogos & derivados , Aminoácido Oxidorreductasas/metabolismo , Ascomicetos/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Cinética , Lisina/síntesis química , Lisina/química , Lisina/farmacología , Estructura Molecular , Relación Estructura-Actividad , Valina/síntesis química , Valina/química , Valina/farmacologíaRESUMEN
Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels.
Asunto(s)
Aspergillus/genética , Glucosa 1-Deshidrogenasa/aislamiento & purificación , Glucosa 1-Deshidrogenasa/metabolismo , Glucosa/metabolismo , Mucor/enzimología , Clonación Molecular , Flavina-Adenina Dinucleótido/metabolismo , Galactosa/metabolismo , Galactosa/farmacología , Expresión Génica , Glucosa/farmacología , Glucosa 1-Deshidrogenasa/química , Glucosa 1-Deshidrogenasa/genética , Glicosilación , Maltosa/metabolismo , Maltosa/farmacología , Peso Molecular , Especificidad por Sustrato/efectos de los fármacos , Xilosa/metabolismo , Xilosa/farmacologíaRESUMEN
Fructosyl peptide oxidase (FPOX) catalyses the oxidation of α-glycated dipeptides such as N(α)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.09, c = 220.48 Å, and that of EFP-T5M belonged to the monoclinic space group P2(1), with unit-cell parameters a = 43.00, b = 230.05, c = 47.27 Å, ß = 116.99°. The crystals of CFP-T7 and EFP-T5M diffracted to 1.8 and 1.6 Å resolution, respectively.
Asunto(s)
Aminoácido Oxidorreductasas/química , Eupenicillium/enzimología , Eurotiales/enzimología , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de PoliacrilamidaRESUMEN
Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50 degrees C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50 degrees C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.
Asunto(s)
Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/genética , Ascomicetos/enzimología , Evolución Molecular Dirigida , Aminoácido Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ascomicetos/química , Ascomicetos/genética , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Mutagénesis , Ingeniería de Proteínas , TemperaturaRESUMEN
Fructosyl peptide oxidase is a flavoenzyme that catalyzes the oxidative deglycation of N-(1-deoxyfructosyl)-Val-His, a model compound of hemoglobin (Hb)A(1C). To develop an enzymatic method for the measurement of HbA(1C), we screened for a proper protease using N-(1-deoxyfructosyl)-hexapeptide as a substrate. Several proteases, including Neutral protease from Bacillus polymyxa, were found to release N-(1-deoxyfructosyl)-Val-His efficiently, however no protease was found to release N-(1-deoxyfructosyl)-Val. Neutral protease also digested HbA(1C) to release N-(1-deoxyfructosyl)-Val-His, and then the fructosyl peptide was detected using fructosyl peptide oxidase. The linear relationship was observed between the concentration of HbA(1C) and the absorbancy of fructosyl peptide oxidase reaction, hence this new method is a practical means for measuring HbA(1C.).
Asunto(s)
Aminoácido Oxidorreductasas/química , Bacillus/enzimología , Proteínas Bacterianas/química , Bioensayo , Endopeptidasas/química , Hemoglobina Glucada/análisis , Dipéptidos/análisis , Humanos , Oxidación-Reducción , Sensibilidad y EspecificidadRESUMEN
Senescence marker protein-30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases androgen-independently with aging. We have now characterized a unique property of SMP30, the hydrolysis of diisopropyl phosphorofluoridate (DFP), which is similar to the chemical warfare nerve agents sarine, soman and tabun. Hydrolysis of DFP was stimulated equally well by 1 mM MgCl2, MnCl2 or CoCl2, to a lesser extent by 1 mM CdCl2 but not at all by 1 mM CaCl2. No 45Ca2+-binding activity was detected for purified SMP30, suggesting that SMP30 is not a calcium-binding protein, as others previously stated. Despite the sequence similarity between SMP30 and a serum paraoxonase (PON), the inability of SMP30 to hydrolyze PON-specific substrates such as paraoxon, dihydrocoumarin, gamma-nonalactone, and delta-dodecanolactone indicate that SMP30 is distinct from the PON family. We previously established SMP30 knockout mice and have now tested DFPase activity in their livers. The livers from wild-type mice contained readily detectable DFPase activity, whereas no such enzyme activity was found in livers from SMP30 knockout mice. Moreover, the hepatocytes of SMP30 knockout mice were far more susceptible to DFP-induced cytotoxicity than those from the wild-type. These results indicate that SMP30 is a unique DFP hydrolyzing enzyme in the liver and has an important detoxification effect on DFP. Consequently, a reduction of SMP30 expression might account for the age-associated deterioration of cellular functions and enhanced susceptibility to harmful stimuli in aged tissue.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Isoflurofato/metabolismo , Hígado/metabolismo , Envejecimiento , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa/sangre , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Hidrolasas de Éster Carboxílico/metabolismo , Muerte Celular , Cloruros/farmacología , Cobalto/farmacología , Cumarinas/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Luciferina de Luciérnaga/metabolismo , Hepatocitos/metabolismo , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular , Lactonas/farmacología , Cloruro de Magnesio/farmacología , Masculino , Compuestos de Manganeso/farmacología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Paraoxon/farmacología , Unión Proteica , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , SulfotransferasasRESUMEN
To develop an enzymatic measurement of HbA(1C), two key enzymes, i.e., fructosyl peptide oxidase and Aspergillus protease were characterized. Fructosyl peptide oxidase from Eupenicillium terrenum was a flavoenzyme that could catalyze the oxidation of N-(1-deoxyfructosyl)-Val-His. The enzyme showed high specificity toward alpha-glycated molecules, therefore it seemed suitable for the HbA(1C) assay. Since high levels of FPOX expression seemed toxic to host cells, we applied a gene expression system using a bacteriophage vector and achieved high levels of expression in Escherichia coli. Next, we found that Aspergillus protease was able to digest N-(1-deoxyfructosyl)-hexapeptide, a glycated peptide that was released from the beta-chain of HbA(1C) by Glu-C endoproteinase. We showed that the N-(1-deoxyfructosyl)-Val-His released from N-(1-deoxyfructosyl)-hexapeptide by Aspergillus protease could be assayed enzymatically using fructosyl peptide oxidase, therefore these enzymes could be applied to the enzymatic measurement of HbA(1C).
Asunto(s)
Pruebas Enzimáticas Clínicas/métodos , Diabetes Mellitus/diagnóstico , Hemoglobina Glucada/análisis , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Aspergillus/enzimología , Bacteriófagos/genética , Cromatografía Líquida de Alta Presión , Endopeptidasas/metabolismo , Escherichia coli/genética , Vectores Genéticos , Hemoglobina Glucada/metabolismo , Glicosilación , Humanos , Cinética , Péptidos/química , Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Fructosyl peptide oxidases, enzymes that are active against a model compound of glycated hemoglobin, N(alpha)-fructosyl valyl-histidine, were characterized. To identify the primary structure of fructosyl peptide oxidases, we have prepared cDNA libraries from Eupenicillium terrenum ATCC18547 and Coniochaeta sp. NISL9330. The coding regions, both fungal fructosyl peptide oxidases consisting of 1314-bp, were obtained with degenerated primers based on the amino acid sequences and specific primers by 3(') and 5(') RACE (rapid amplification of cDNA ends). By their sequence similarities and substrate specificities, fructosyl peptide oxidases and their homologs could be categorized into two groups: (A) enzymes that preferably oxidize alpha-glycated molecules and (B) enzymes that preferably oxidize epsilon-glycated molecules. We showed that recombinant fructosyl peptide oxidases could be used to detect protease-treated fructosyl-hexapeptide, a glycated peptide that is released from HbA(1C) by endoproteinase Glu-C, suggesting these enzymes could be useful for the enzymatic measurement of HbA(1C).
Asunto(s)
Aminoácido Oxidorreductasas/biosíntesis , Aminoácido Oxidorreductasas/química , Ascomicetos/química , Ascomicetos/enzimología , Hemoglobina Glucada/análisis , Hemoglobina Glucada/química , Técnicas de Sonda Molecular , Aminoácido Oxidorreductasas/clasificación , Aminoácido Oxidorreductasas/aislamiento & purificación , Secuencia de Aminoácidos , Ascomicetos/clasificación , Clonación Molecular , Activación Enzimática , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia , Especificidad por SustratoRESUMEN
Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N(alpha)-fructosyl valyl-histidine (alpha-keto-amine) and N(epsilon)-fructosyl lysine (epsilon-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both alpha-keto-amine and epsilon-keto-amine, and (2) enzymes that preferably oxidize alpha-keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric ( M(r)=50,000), was most active at 40 degrees C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K(m) values for N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine were 2.30 and 1.69 mM, respectively. N(alpha)-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A(1C), the N-terminal valine residue of the beta-subunit of which is glycated.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Aminoácido Oxidorreductasas/metabolismo , Hemoglobina Glucada/análogos & derivados , Lisina/análogos & derivados , Sordariales/enzimología , Valina/análogos & derivados , Cromatografía Líquida de Alta Presión , Hemoglobina Glucada/metabolismo , Histidina/análogos & derivados , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Lisina/metabolismo , Especificidad por Sustrato , Temperatura , Valina/metabolismoRESUMEN
In the firefly light organ, oxyluciferin, a product of the light-emitting reaction of firefly luciferase, is thought to be converted into luciferin. Previously, we isolated the luciferin-regenerating enzyme (LRE) from Photinus pyralis. LRE plays an important role in the recycling of oxyluciferin into luciferin. We have cloned two cDNAs encoding LRE, G-LRE and H-LRE, from poly(A)+ RNA of the lanterns of Luciola cruciata and Luciola lateralis, using reverse transcription-polymerase chain reaction, 5'-RACE (5'-rapid amplification of cDNA ends) and 3'-RACE. The putative translation products have molecular masses of 33,804 and 34,285 Da, corresponding to 309 and 307 amino acids, respectively. The deduced amino acid sequence of G-LRE shows 57 and 56% identity with H-LRE and A-LRE (P. pyralis), respectively. LRE (G-LRE, H-LRE, A-LRE) shows at most 39% amino acid sequence identity with insect anterior fat protein (AFP) and mammalian senescence marker protein-30 (SMP30). G-LRE and H-LRE were successfully expressed under the control of the lac promoter in Escherichia coli.
Asunto(s)
Escarabajos/genética , ADN Complementario/genética , Indoles , Proteínas de Insectos/genética , Ligasas/genética , Pirazinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escarabajos/enzimología , ADN Complementario/química , Escherichia coli/genética , Luciferina de Luciérnaga/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas de Insectos/metabolismo , Ligasas/metabolismo , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
To improve the practical usefulness of the firefly luciferase, we performed gene chimerization between Photinus pyralis luciferase and a thermostable variant of Luciola cruciata luciferase. One chimeric luciferase showed low K(m) value for substrate ATP and similar stability to thermostable L. cruciata luciferase. We then introduced random mutations in the corresponding gene and screened for increased catalytic efficiency. Amino acid replacement of Thr219, Val239 and Val290 affected the kinetic parameters. Therefore, we combined these three mutations. One mutant, ABcT219I,V239I, showed high catalytic efficiency comparable to P. pyralis luciferase and high stability similar to thermostable L. cruciata luciferase. The pH-dependence of the bioluminescence emission spectra was also examined. In contrast to wild-type firefly luciferases characterized to date, the mutant did not show the pH-dependent red spectrum shift.
Asunto(s)
Escarabajos/enzimología , Escarabajos/genética , Luciferasas/química , Luciferasas/genética , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Animales , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Luciferasas/metabolismo , Mediciones Luminiscentes , Estructura Molecular , Mutagénesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TemperaturaRESUMEN
Agrobacterium tumefaciens AgaE-like protein had a similar sequence to that of a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1. To characterize the AgaE-like protein, we produced the enzyme in Escherichia coli, and purified it to homogeneity. The molecular mass of recombinant AgaE-like protein was 42 kDa on SDS-PAGE and 85 kDa on gel filtration. The protein acted on N-fructosyl valine and N-fructosyl glycine as substrates, but not on glycated protein or N(epsilon)-fructosyl lysine. Apparent Km for N-fructosyl valine and N-fructosyl glycine were 1.64 and 0.31 mM, respectively. The AgaE-like protein had maximum activity at pH 7.8 and 35 degrees C in 0.1 M potassium phosphate, but more than 80% of its activity was lost at 40 degrees C or more. In contrast to eukaryotic fructosyl amino acid oxidases, the AgaE-like protein contained noncovalently bound FAD as a cofactor and was inactive against N(epsilon)-fructosyl N(alpha)-Z(benzyloxycarbonyl)-lysine. These characteristics were similar to a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1, suggesting that these prokaryotic enzymes comprise a new family of fructosyl amino acid oxidases.