RESUMEN
Contaminating proteins have been identified by "shotgun" proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas de Escherichia coli/aislamiento & purificación , Flavoproteínas/aislamiento & purificación , Hemoproteínas/aislamiento & purificación , Proteómica/métodos , Secuencia de Aminoácidos , Catalasa/aislamiento & purificación , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Proteínas de Choque Térmico/aislamiento & purificación , Hemoproteínas/genética , Hemoproteínas/metabolismo , Humanos , Factor Tu de Elongación Peptídica/aislamiento & purificación , Isomerasa de Peptidilprolil/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/aislamiento & purificación , Sefarosa/análogos & derivados , Sefarosa/químicaRESUMEN
Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.
Asunto(s)
Proteínas Fúngicas/aislamiento & purificación , Glucosamina/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Plásmidos/metabolismo , Saccharomyces cerevisiae/genética , Sporothrix/química , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Fumaratos/química , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expresión Génica , Prueba de Complementación Genética , Biblioteca Genómica , Glucosamina/análogos & derivados , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Cinética , Sistemas de Lectura Abierta , Plásmidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Sporothrix/enzimología , beta-Alanina/análogos & derivados , beta-Alanina/químicaRESUMEN
Rationally designed muteins of Candida albicans glucosamine-6-phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versions containing internal oligoHis fragments were constructed: (i) by substituting residues 343-348 of the interdomain undecapeptide linker with hexaHis, (ii) by replacing solvent-exposed residues 655-660 of the isomerase domain with hexaHis, and (iii) by replacing amino acids at positions 568 and 569 with His residues to generate the three-dimensional hexaHis microdomain in the enzyme quaternary structure. The resulting constructs were effectively purified to near homogeneity by rapid, one-step immobilized metal-ion affinity chromatography and demonstrated activity and catalytic properties comparable with that of the wild-type enzyme. The construct containing the 655-660 hexaHis insert was found to be a homodimeric protein, which is the first reported example of such quaternary structure of glucosamine-6-phosphate synthase of eukaryotic origin.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Biocatálisis , Candida albicans/química , Cromatografía de Afinidad , Escherichia coli/genética , Proteínas Fúngicas/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Cinética , Modelos Moleculares , Mutación , Ingeniería de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
N(3)-Oxoacyl derivatives of L-2,3-diaminopropanoic acid 1-4, containing either an epoxide group or a conjugated double bond system, inactivate Saccharomyces cerevisiae glucosamine-6-phosphate (GlcN-6-P) synthase in a time- and concentration dependent manner. The results of kinetics studies on inactivation suggested a biphasic course, with formation of the enzyme-ligand complex preceding irreversible modification of the enzyme. The examined compounds differed markedly in their affinity to the enzyme active site. Inhibitors containing a phenyl ketone moiety bound much more strongly than their methyl ketone counterparts. The molecular mechanism of enzyme inactivation by phenyl ketone compounds 1 and 3 was elucidated by using a stepwise approach with 2D NMR, MS and UV-visible spectroscopy. A substituted thiazine derivative was identified as the final product of a model reaction between an epoxide compound, 1, and L-cysteine ethyl ester (CEE); and the respective cyclic product, found as a result of reaction between 1 and CGIF tetrapeptide, was identical to the N-terminal fragment of GlcN-6-P synthase. On the other hand, the reaction of a double-bond-containing compound, 3, with CEE, CGIF and GlcN-6-P synthase led to the formation of a C-S bond, without any further conversion or rearrangement. Molecular mechanisms of the reactions studied are proposed.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , beta-Alanina/análogos & derivados , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Modelos Moleculares , Estructura Molecular , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , beta-Alanina/síntesis química , beta-Alanina/química , beta-Alanina/farmacologíaRESUMEN
The first committed step of the biosynthetic pathway leading to uridine-5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is catalyzed by glucosamine-6-phosphate synthase (GlcN-6-P synthase), an enzyme proposed as a potential antifungal chemotherapy target. Here, we describe the purification and biochemical characterization of the native enzyme from the dimorphic pathogenic fungus Sporothrix schenckii. The availability of the pure protein facilitated its biochemical characterization. The enzyme exhibited subunit and native molecular masses of 79 and 350+/-5 kDa, respectively, suggesting a homotetrameric structure. Isoelectric point was 6.26 and K(m) values for fructose-6-phosphate and L-glutamine were 1.12+/-0.3 and 2.2+/-0.7 mM, respectively. Inhibition of activity by UDP-GlcNAc was enhanced by Glc-6-P and phosphorylation stimulated GlcN-6-P synthase activity without affecting the enzyme sensitivity to the aminosugar. A glutamine analogue, FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid] was a more potent inhibitor of activity than ADMP (2-Amino-2-deoxy-D-mannitol-6-phosphate) but the latter was a stronger inhibitor of growth in two culture media. To our knowledge, this is the first report on the purification and biochemical characterization of a non-recombinant GlcN-6-P synthase from a true dimorphic fungus. Inhibition of enzyme activity and fungal growth by specific inhibitors of GlcN-6-P synthase strongly reinforces the role of this enzyme as a potential target for antifungal chemotherapy.
Asunto(s)
Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Sporothrix/enzimología , Inhibidores Enzimáticos/metabolismo , Fructosafosfatos/metabolismo , Fumaratos/metabolismo , Proteínas Fúngicas/química , Glutamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Punto Isoeléctrico , Cinética , Manosa/análogos & derivados , Manosa/metabolismo , Peso Molecular , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/química , Uridina Difosfato N-Acetilglucosamina/metabolismo , beta-Alanina/análogos & derivados , beta-Alanina/metabolismoRESUMEN
Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.
Asunto(s)
Medios de Cultivo/química , Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animales , Cromatografía de Afinidad , Endotoxinas/biosíntesis , Endotoxinas/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/biosíntesis , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Microbiología Industrial/métodos , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Human L-glutamine: D-fructose-6-phosphate amidotransferase (Gfat1), a recognized target in type 2 diabetes complications, was expressed in Sf9 insect cells with an internal His(6)-tag and purified to homogenity. Two different microplate assays that quantify, respectively D-glucosamine-6-phosphate and L-glutamate were used to analyze the enzyme kinetic properties. The recombinant human L-glutamine: D-fructose-6-phosphate amidotransferase isoform 1 exhibits Michaelis parameters K(m)(Fru-6P)=0.98 mM and K(m)(Gln)=0.84 mM which are similar to the values reported for the same enzyme from different sources. The stimulation of hydrolysis of the alternate substrate L-glutamine para-nitroanilide by D-fructose-6P (Fru-6P) afforded a K(d) of 5 microM for Fru-6P.
Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/biosíntesis , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Histidina/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Animales , Células Cultivadas , Dicroismo Circular , Clonación Molecular , Activación Enzimática , Fructosafosfatos/química , Ácido Glutámico/química , Glutaminasa/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Humanos , Hidrólisis , Insectos/citología , Insectos/metabolismo , Cinética , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
Expression plasmids containing recombinant genes encoding three His(6)-tagged versions of the enzyme, glucosamine-6-phosphate synthase from Candida albicans, were constructed and overexpressed in Escherichia coli. The gene products were purified by metal-affinity chromatography to near homogeneity with 77-80% yield and characterized in terms of size and enzymatic properties. Presence of oligohistidyl tags at either of two ends did not affect enzyme quarternary structure but strongly influenced its catalytic activity. The His6-N-tagged enzyme completely lost an ability of glucosamine-6-phosphate formation and amidohydrolase activity but retained the hexosephosphate-isomerising activity. On the other hand, two His6-C-tagged versions of glucosamine-6-phosphate synthase exhibited amidohydrolase activity almost equal to that of the wild-type enzyme but only 18% of its hexosephosphate-isomerising activity and about 1.5% of the synthetic activity.
Asunto(s)
Candida albicans/enzimología , Escherichia coli , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Candida albicans/genética , Catálisis , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/biosíntesis , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Estructura Cuaternaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Novel inhibitors 1-4 of glucosamine-6-phosphate synthase from Candida albicans have been designed based on acylation of the N3 amino group of L-2,3-diaminopropanoic acid with the corresponding ketoacids. These inhibitors have been shown to alkylate the fungal enzyme in a time-dependent manner. Compound 3 containing trans-beta-benzoyl acrylic acid as an acyl residue was found to be the most potent inhibitor in the series. Dipeptides composed of the active inhibitors and norvaline demonstrated potent antifungal activity against selected strains of Candida spp. and Saccharomyces cerevisiae. Their activity was reversed upon addition of N-acetylglucosamine to the medium.
Asunto(s)
Antifúngicos/farmacología , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , beta-Alanina/análogos & derivados , beta-Alanina/farmacología , Alquilación , Antifúngicos/síntesis química , Sitios de Unión , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Dipéptidos/síntesis química , Inhibidores Enzimáticos/síntesis química , Fructosafosfatos/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Modelos Moleculares , beta-Alanina/síntesis químicaRESUMEN
The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).
Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hígado/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Disulfuros/farmacología , Ditiotreitol/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The Candida albicans GFA1 gene encoding glucosamine-6-phosphate synthase, an enzyme of cell wall biosynthesis pathway in fungi and bacteria, recently an object of interest as a target for the chemotherapy of systemic mycoses, was PCR amplified and cloned to an Escherichia coli expression vector pET23b. The activity of the enzyme in the lysates from the overproducing E. coli strain was approximately 50-100 times higher than in the lysates from the control E. coli strain. This abundant overproduction allows to purify milligram amounts of the enzyme to homogeneity.
Asunto(s)
Candida albicans/genética , Escherichia coli/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Candida albicans/enzimología , Precipitación Química , Cromatografía , Clonación Molecular , ADN de Hongos/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Expresión Génica , Genes Fúngicos , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited Km(Gln) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with kcat = 1150 min-1. The isoelectric point of 4.6 +/- 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with nH = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1. 21 +/- 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast --> mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Animales , Northern Blotting , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Focalización Isoeléctrica , Cinética , Peso Molecular , Conformación Proteica , Proteínas Recombinantes , Mapeo Restrictivo , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. N-Iodoacetylglucosamine 6-phosphate is an active site-directed irreversible inactivator of GlmS from Escherichia coli (kinact/KI = 17 (+/-3) m-1 s-1). Both fructose 6-phosphate and glutamine protect the enzyme from inactivation, indicating that this reagent is directed at both the sugar binding site and the glutamine binding site. Protection studies with fructose 6-phosphate demonstrate that the value of the dissociation constant for fructose 6-phosphate is 3.3 (+/-0.5) x 10(-7) m, approximately 3 orders of magnitude less than the Kia value for this substrate determined from initial velocity experiments (Badet, B., Vermoote, P., and Le Goffic, F. (1988) Biochemistry 27, 2282-2287).
Asunto(s)
Acetilglucosamina/farmacología , Escherichia coli/enzimología , Fructosafosfatos/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Acetilglucosamina/análogos & derivados , Acetilglucosamina/síntesis química , Sitios de Unión , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Cinética , Matemática , Modelos Teóricos , Estructura MolecularRESUMEN
Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.
Asunto(s)
ADN/genética , Escherichia coli/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity. Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label [2-3H]-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function. CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity. These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain.
Asunto(s)
Escherichia coli/enzimología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Marcadores de Afinidad , Amidohidrolasas/química , Amidohidrolasas/aislamiento & purificación , Sitios de Unión , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/aislamiento & purificación , Fructosafosfatos , Glutamina , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Peso Molecular , Péptido HidrolasasRESUMEN
L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.
Asunto(s)
Escherichia coli/enzimología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Glutamina/metabolismo , Transaminasas/aislamiento & purificación , Aminoácidos/análisis , Sitios de Unión , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Cinética , Peso Molecular , Unión Proteica , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
Synthetic derivatives of N3-fumaroyl-L-2,3-diaminopropanoic acid constitute the novel group of glutamine analogs. They are powerful, competitive inhibitors of the glucosamine synthetase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring), EC 5.3.1.19) from Candida albicans with respect to glutamine and uncompetitive with respect to D-fructose 6-phosphate. Some of the compounds tested irreversibly inactivate glucosamine synthetase with Kinact values of 10(-4) to 10(-6) M. The addition of glutamine protects enzyme from the inactivation, while the absence of D-fructose 6-phosphate lowers the rate of inactivation. An ordered, sequential mechanism is suggested for binding of the inhibitors to the glutamine-binding site. A number of tested compounds act as active-site-directed, irreversible inhibitors. It is suggested that derivatives of N3-fumaroyl-L-2,3-diaminopropanoic acid should be classified as mechanism-based enzyme inactivators. Structural requirements for an effective inactivator containing N3-fumaroyl-L-2,3-diaminopropanoic acid moiety are discussed.
Asunto(s)
Alanina/análogos & derivados , Candida albicans/enzimología , Carbohidrato Epimerasas/antagonistas & inhibidores , Fumaratos/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , beta-Alanina/análogos & derivados , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Cinética , Relación Estructura-Actividad , beta-Alanina/farmacologíaAsunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Hígado/enzimología , Transaminasas/aislamiento & purificación , Amoníaco/metabolismo , Animales , Sitios de Unión , Cromatografía/métodos , Cromatografía DEAE-Celulosa/métodos , Clonación Molecular , Durapatita , Glutamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hidroxiapatitas , Cinética , RatasRESUMEN
A glucosamine-P isomerase has been identified in Proteus mirabilis. The 113-fold purified enzyme exhibits a pH optimum of 7.5 with a secondary maximum at 8.5 and a temperature optimum at 37 degrees C. The apparent Km was 13.3 mM for fructose-6-P and 18.8 mM for L-glutamine. Molecular weight of the enzyme has been estimated as 120 000 and the protein can be dissociated in four subunits by SDS-polyacrylamide electrophoresis.