RESUMEN
Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.
Asunto(s)
Apoenzimas/biosíntesis , Arthrobacter/enzimología , Hidrolasas/biosíntesis , Apoenzimas/química , Apoenzimas/genética , Simulación por Computador , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/metabolismo , Herbicidas/metabolismo , Hidrolasas/química , Hidrolasas/genética , Hidrólisis , Cinética , Modelos Moleculares , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Missense , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidad , Triazinas/metabolismoRESUMEN
The iron-molybdenum cofactor (the M-cluster) serves as the active site of molybdenum nitrogenase. Arguably one of the most complex metal cofactors in biological systems, the M-cluster is assembled through the formation of an 8Fe core prior to the insertion of molybdenum and homocitrate into this core. Here, we review the recent progress in the research area of M-cluster assembly, with an emphasis on our work that provides useful insights into the mechanistic details of this process.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Molibdoferredoxina/biosíntesis , Nitrogenasa/biosíntesis , Apoenzimas/biosíntesis , Apoenzimas/química , Proteínas Bacterianas/química , Dominio Catalítico , Coenzimas/biosíntesis , Coenzimas/química , Modelos Moleculares , Molibdoferredoxina/química , Nitrogenasa/química , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
The H-cluster is a complex bridged metal assembly at the active site of [FeFe]-hydrogenases that consists of a [4Fe-4S] subcluster bridged to a 2Fe-containing subcluster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, and HydG) responsible for the biosynthesis of the H-cluster and the maturation of active [FeFe]-hydrogenase have previously been identified and shown to be required for the heterologous expression of active [FeFe]-hydrogenase [Posewitz, M. C., et al. (2004) J. Biol. Chem. 279, 25711-25720]. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subcluster. The spectroscopic and biochemical characterization of HydA(DeltaEFG) (the [FeFe]-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a [4Fe-4S] cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 +/- 0.5 Fe atoms per HydA(DeltaEFG)) and S(2-) (1.8 +/- 0.5 S(2-) atoms per HydA(DeltaEFG)) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S = (1)/(2) EPR signal (g = 2.04 and 1.91) characteristic of a reduced [4Fe-4S](+) cluster. Mossbauer spectroscopic characterization of (57)Fe-enriched HydA(DeltaEFG) provided further evidence of the presence of a redox active [4Fe-4S](2+/+) cluster. Iron K-edge EXAFS data provided yet further support for the presence of a [4Fe-4S] cluster in HydA(DeltaEFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(DeltaEFG) can be activated by the specific maturases only when a [4Fe-4S] cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subcluster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Hidrogenasas/química , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Absorciometría de Fotón , Animales , Apoenzimas/biosíntesis , Apoenzimas/química , Apoenzimas/genética , Biocatálisis , Cloruros , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Compuestos Férricos/química , Análisis de Fourier , Hidrogenasas/biosíntesis , Hidrogenasas/genética , Hierro/química , Proteínas Hierro-Azufre/biosíntesis , Proteínas Hierro-Azufre/genética , Cinética , Modelos Químicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Espectrofotometría Ultravioleta , Espectroscopía de Mossbauer , Sulfuros/químicaRESUMEN
Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.
Asunto(s)
Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Galactoquinasa/genética , Galactoquinasa/aislamiento & purificación , Regulación de la Expresión Génica Arqueal , Pyrococcus horikoshii/enzimología , Apoenzimas/biosíntesis , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/aislamiento & purificación , Proteínas Arqueales/biosíntesis , Proteínas Arqueales/química , Cristalización , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Galactoquinasa/biosíntesis , Galactoquinasa/química , Cinética , Pyrococcus horikoshii/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
An overexpression system for spinach apocytochrome b(6) as a fusion protein to a maltose-binding protein in Escherichia coli was established using the expression vector pMalp2. The fusion of the cytochrome b(6) to the periplasmic maltose-binding protein directs the cytochrome on the Sec-dependent pathway. The cytochrome b(6) has a native structure in the bacterial cytoplasmic membrane with both NH(2) and COOH termini on the same, periplasmic side of the membrane but has the opposite orientation compared to that in thylakoid. Our data also show that in the E. coli cytoplasmic membrane, apocytochrome b(6) and exogenic hemes added into a culture media spontaneously form a complex with similar spectroscopic properties to native cytochrome b(6). Reconstituted membrane-bound cytochrome b(6) contain two b hemes (alpha band, 563 nm; average E(m,7) = -61 +/- 0.84 and -171 +/- 1.27 mV).
Asunto(s)
Citocromos b6/metabolismo , Citoplasma/enzimología , Proteínas de Escherichia coli/metabolismo , Membranas Intracelulares/enzimología , Proteínas de la Membrana/metabolismo , Tilacoides/enzimología , Apoenzimas/biosíntesis , Apoenzimas/genética , Proteínas Portadoras/metabolismo , Frío , Citocromos b6/biosíntesis , Citocromos b6/genética , Citoplasma/metabolismo , Electroquímica , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Hemo/análisis , Hemo/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Unión a Maltosa , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Oxidación-Reducción , Plásmidos , Espectrofotometría , Spinacia oleracea , Coloración y Etiquetado , Tilacoides/metabolismoRESUMEN
The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.
Asunto(s)
Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/metabolismo , Apoenzimas/biosíntesis , Apoenzimas/genética , Apoenzimas/aislamiento & purificación , Apoenzimas/metabolismo , Bacillus/enzimología , Bacillus/genética , Sitios de Unión , Cisteína/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Flavina-Adenina Dinucleótido/síntesis química , Flavina-Adenina Dinucleótido/aislamiento & purificación , Cinética , Mutagénesis , Oxidorreductasas N-Desmetilantes/biosíntesis , Oxidorreductasas N-Desmetilantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sarcosina-Oxidasa , EspectrofotometríaRESUMEN
The increase in Candida albicans infections is caused by the increase in therapies resulting in immunocompromised patients. One factor required for C. albicans pathogenicity is the morphological transition from yeast to hypha. The protein profiles of whole extracts from yeasts and hyphae were examined using two-dimensional electrophoresis to identify the proteins related to the morphological transition. Over 900 protein spots were visualized by silver staining and 11 spots were increased more than three-fold reproducibly during hyphal differentiation. Six of the 11 spots were identified by peptide mass fingerprints, of which three represented PRA1, two PHR1 and the last TSA1. Vertical streak patterns of Pra1p and Phr1p indicated that post-translational modifications seem to be caused by variable glycosylation. Comparative proteome analysis between the wild-type and the deletion mutants, CAMB43 (deltapra1) and CAS10 (deltaphr1), further confirmed the identity of PRA1 and PHR1. Interestingly, Pra1p was downregulated in phr1-deleted mutants. Only PHR1 transcription was increased, indicating that PRA1 and TSA1 are controlled at the post-translational level.
Asunto(s)
Candida albicans/metabolismo , Proteínas Fúngicas/biosíntesis , Hifa/metabolismo , Glicoproteínas de Membrana , Proteínas de Neoplasias , Apoenzimas/biosíntesis , Apoenzimas/genética , Northern Blotting , Candida albicans/citología , Candida albicans/genética , Desoxirribodipirimidina Fotoliasa/biosíntesis , Desoxirribodipirimidina Fotoliasa/genética , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/genética , Expresión Génica , Hifa/citología , Hifa/genética , Mapeo Peptídico , Peroxidasas/biosíntesis , Peroxidasas/genética , Peroxirredoxinas , Proteómica/métodos , ARN de Hongos/genética , ARN de Hongos/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Three key steps of cytochrome c biogenesis in many Gram-negative bacteria, the uptake of heme by the heme chaperone CcmE, the covalent attachment of heme to CcmE, and its subsequent release from CcmE to an apocytochrome c, have been achieved in vitro. apo-CcmE from Escherichia coli preferentially bound to ferric, with high affinity (K(d), 200 nM), rather than ferrous heme. The preference for ferric heme was confirmed by competition with 8-anilino-1-naphthalenesulfonate, which bound to a hydrophobic pocket in apo-CcmE. Reduction under certain conditions of the ferric heme-CcmE complex, which has characteristics of a b-type cytochrome, resulted in covalent attachment of heme to the protein. The resulting in vitro-produced holo-CcmE was identical to the in vivo-produced holo-CcmE, proving that unmodified Fe-protoporphyrin IX is incorporated into CcmE. Only noncovalent binding of mesoheme to CcmE was observed, thus implicating at least one vinyl group in covalent binding of heme to CcmE. Heme transferred in vitro from holo-CcmE to apocytochrome c, provided the heme was reduced. The necessity for reduced holo-CcmE might explain the role of the heme chaperone, i.e., prevention of reaction of ferric heme with apocytochrome and thus avoidance of incorrect side products. In addition, an AXXAH mutant of the CXXCH binding motif in the apocytochrome c was unable to accept heme from holo-CcmE. These in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during c-type cytochrome maturation in many species of bacteria in vivo.
Asunto(s)
Apoproteínas/biosíntesis , Proteínas de la Membrana Bacteriana Externa/metabolismo , Grupo Citocromo c/biosíntesis , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Hemo/metabolismo , Hemoproteínas/metabolismo , Apoenzimas/biosíntesis , Apoproteínas/metabolismo , Grupo Citocromo c/genética , Cartilla de ADN , Escherichia coli/genética , CinéticaRESUMEN
The alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi. The alpha-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The LYS2 and LYS5 genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of Candida albicans, a pathogenic yeast, in the presence of CoA and C. albicans lys2 mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type C. albicans CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of C. albicans.
Asunto(s)
Aldehído Oxidorreductasas/biosíntesis , Candida albicans/enzimología , Lisina/biosíntesis , Panteteína/análogos & derivados , Procesamiento Proteico-Postraduccional , Aldehído Oxidorreductasas/genética , Secuencia de Aminoácidos , Apoenzimas/biosíntesis , Candida albicans/genética , Coenzima A/metabolismo , Secuencia Conservada , Activación Enzimática , Holoenzimas/biosíntesis , L-Aminoadipato-Semialdehído Deshidrogenasa , Panteteína/metabolismoRESUMEN
Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study investigates the influence of endogenously generated NO (or NO-derived species) on the relative expression of cytochrome P450 (CYP) isoforms in rat hepatocytes. Our results support the view that loss of P450 holoenzyme in culture is the ultimate consequence of a NO driven process, activated during the common hepatocyte isolation procedure, that leads to an accelerated and selective degradation of specific CYP apoproteins. Under conditions in which NO and peroxynitrite formation is operative, changes in the level of specific CYP isoforms result in a significant alteration of the CYP apoprotein profile that after 24 h of culture is quite different from that found in the liver of uninduced rats. This process is reverted by the early and efficient inhibition of NO synthesis, which allows for (1) maintenance of total P450 holoenzyme content, (2) preservation of the initial constitutive CYP pattern in culture and (3) the early expression of the normal inducibility in response to model inducers.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/metabolismo , Óxido Nítrico/metabolismo , Animales , Apoenzimas/biosíntesis , Apoenzimas/metabolismo , Western Blotting , Células Cultivadas , Sistema Enzimático del Citocromo P-450/biosíntesis , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Holoenzimas/metabolismo , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , beta-naftoflavona/farmacologíaRESUMEN
Characterization of a series of urease-negative transposon mutations of Actinobacillus pleuropneumoniae revealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF, apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. The cbiM and cbiQ genes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and the cbiO gene encodes a cobalt transport ATP-binding protein homologue. The product of the cbiK gene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein. Escherichia coli clones containing this putative transport operon together with the urease genes of A. pleuropneumoniae were urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl(2) for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF, utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function in A. pleuropneumoniae remains to be determined.
Asunto(s)
Actinobacillus pleuropneumoniae/genética , Genes Bacterianos , Proteínas de Transporte de Membrana/genética , Níquel/metabolismo , Ureasa/biosíntesis , Actinobacillus pleuropneumoniae/enzimología , Actinobacillus pleuropneumoniae/patogenicidad , Animales , Apoenzimas/biosíntesis , Transporte Biológico , Cobalto/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Insercional , Sistemas de Lectura Abierta , Pleuroneumonía/etiología , Pleuroneumonía/veterinaria , Análisis de Secuencia de ADN , Porcinos , Enfermedades de los Porcinos/etiologíaRESUMEN
C-type cytochromes from various sources show substantial structural conservation. For the covalent attachment of heme groups to apocytochromes, however, three different enzyme systems have been described so far. We have examined the ability of the heme ligation systems of Escherichia coli and of Saccharomyces cerevisiae to process cytochromes from S. cerevisiae, Paracoccus denitrificans, and Synechocystis sp. PCC 6803. E. coli's maturation system with at least eight different proteins accepted all these cytochromes for heme ligation. The single subunit heme lyase from S. cerevisiae mitochondria, on the other hand, failed to attach heme groups to cytochromes of prokaryotic origin.
Asunto(s)
Proteínas Bacterianas , Grupo Citocromo c/genética , Citocromos c , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Apoenzimas/biosíntesis , Apoproteínas/biosíntesis , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Sitios de Unión , Grupo Citocromo c/biosíntesis , Escherichia coli/metabolismo , Hemoproteínas/biosíntesis , Liasas/biosíntesis , Proteínas de la Membrana/biosíntesis , Plásmidos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.
Asunto(s)
Apoenzimas/biosíntesis , Catalasa/biosíntesis , Escherichia coli/genética , Holoenzimas/biosíntesis , Thermus/enzimología , Línea Celular , Cromatografía en Gel , Activación Enzimática , Chaperonas Moleculares/biosíntesisRESUMEN
The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH7 and pH8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.
Asunto(s)
Sustitución de Aminoácidos , Membrana Celular/enzimología , Escherichia coli/enzimología , Glucosa Deshidrogenasas/metabolismo , Histidina/genética , Tirosina/genética , Apoenzimas/biosíntesis , Apoenzimas/química , Apoenzimas/aislamiento & purificación , Apoenzimas/metabolismo , Sitios de Unión , Transporte de Electrón , Escherichia coli/citología , Escherichia coli/genética , Glucosa Deshidrogenasas/química , Glucosa Deshidrogenasas/genética , Glucosa Deshidrogenasas/aislamiento & purificación , Hexosas/química , Hexosas/metabolismo , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Magnesio/metabolismo , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Oxígeno/metabolismo , Cofactor PQQ , Pentosas/química , Pentosas/metabolismo , Quinolonas/metabolismo , Quinonas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.
Asunto(s)
Acetil-CoA Carboxilasa/química , Escherichia coli/enzimología , Acetil-CoA Carboxilasa/biosíntesis , Acetil-CoA Carboxilasa/metabolismo , Secuencia de Aminoácidos , Apoenzimas/biosíntesis , Apoenzimas/química , Apoenzimas/metabolismo , Biotina/biosíntesis , Isótopos de Carbono , Cristalografía por Rayos X , Holoenzimas/biosíntesis , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , SolucionesRESUMEN
The flavoenzyme d-aspartate oxidase from beef kidney (DASPO, EC 1.4. 3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9-20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of -97 mV and -157 mV were determined for free and l-(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25 degrees C. The large positive shift in the redox potential of the coenzyme compared to free FAD (-207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d-aspartatic acid, d-glutamic acid, N-methyl-d-aspartic acid, d,l-cysteic acid, d-homocysteic acid, d, l-2-amino-3-phosphonopropanoic acid, d-alpha-aminoadipic acid, d-aspartic acid-beta-hydroxamate, glycyl-d-aspartic acid and cis-2, 3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25 degrees C are reported.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Riñón/enzimología , Aminoácido Oxidorreductasas/biosíntesis , Animales , Apoenzimas/biosíntesis , Bovinos , D-Aspartato Oxidasa , Escherichia coli/metabolismo , Expresión Génica , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Ethanol, acetone, diet and starvation, known modulators of the hepatic cytochrome P450 (CYP)-dependent microsomal monooxygenase system, were assessed for their effects on cytochrome P450 isozyme content and monooxygenase activities in the male rat kidney. In acute experiments, rats were either treated with acetone, fasted or given a combination of the two treatments. Acetone treatment alone induced CYP2E1-dependent p-nitrophenol hydroxylase activity in kidney microsomes by 8-fold. This was accompanied by a 6-fold increase in CYP2E1 apoprotein as determined by Western blot analysis. There was, however, no significant increase in steady-state levels of CYP2E1 mRNA as measured by Northern blot analysis. Starvation also induced CYP2E1 apoprotein in the kidney and, as has been reported previously in the liver, had a synergistic inductive effect with acetone. CYP2B and CYP3A apoproteins were also induced by acetone, starvation and starvation/acetone combinations in the kidney. Immunohistochemical analysis revealed localization of CYP2E1 and CYP2B principally in the cortex associated with tubular cells. This distribution was maintained upon starvation/acetone treatment. Two induction experiments were performed in which the ethanol was administered as part of a system of total enteral nutrition (TEN). A short-term study was conducted in which ethanol was administered for 8 days in two liquid diets of different composition, and a chronic experiment was performed in which ethanol was administered for 35 days. A diet-independent 6-fold increase in CYP2E1 apoprotein was observed in the short-term experiment. Expression of CYP3A and CYP2A cross-reactive apoproteins in kidney microsomes appeared to be affected by alterations in diet but, were unaffected by ethanol treatment. In the chronic 35-day ethanol exposure experiment, CYP2E1 apoprotein was also elevated 6-fold and this was found to be accompanied by a significant 3-fold increase in CYP2E1 mRNA. In the same study, no ethanol effects were apparent on expression of CYP2B and CYP3A apoproteins. Thus, acetone induced a variety of renal cytochrome P450 forms in addition to CYP2E1, while ethanol appeared to be a much more specific renal CYP2E1 inducer. Furthermore, as reported in the liver, acetone and ethanol appeared to induce CYP2E1 in the kidney by different mechanisms.
Asunto(s)
Acetona/farmacología , Alcoholismo/enzimología , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Regulación Enzimológica de la Expresión Génica , Riñón/enzimología , Microsomas/enzimología , Animales , Apoenzimas/biosíntesis , Citocromo P-450 CYP2E1/biosíntesis , Citocromo P-450 CYP3A , Dieta , Nutrición Enteral , Etanol/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Oxidorreductasas N-Desmetilantes/biosíntesis , Ratas , Ratas Sprague-Dawley , InaniciónRESUMEN
In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast. Here we report the identification of the UME6 gene of S. cerevisiae as a regulator of UAS(PHR1) activity. Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene. Surprisingly, the effect of deletion of UME6 is growth phase dependent. In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift. Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant. Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site. Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.
Asunto(s)
Apoenzimas/genética , Proteínas de Ciclo Celular , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Endodesoxirribonucleasas , Regulación Fúngica de la Expresión Génica , Glicoproteínas de Membrana , Proteínas Serina-Treonina Quinasas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Apoenzimas/biosíntesis , Sitios de Unión , Quinasa de Punto de Control 2 , Clonación Molecular , Desoxirribodipirimidina Fotoliasa/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Dosificación de Gen , Genes Fúngicos , Mutagénesis , Unión Proteica , Proteínas Quinasas/genética , Dímeros de Pirimidina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/efectos de la radiación , Transcripción Genética , Rayos Ultravioleta/efectos adversosRESUMEN
The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein, Escherichia coli thioredoxin (Trx), a protein that does not naturally contain a transition metal-binding site. Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site. The electronic spectra of the holoprotein and its N3- and F- adducts are analogous to those previously reported for native {Fe3+}SOD. Activity assays showed that {Fe3+}Trx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type E. coli iron SOD indicated that {Fe3+}Trx-SOD catalyzes the dismutation reaction at a rate on the order of 10(5) M-1s -1. The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.
Asunto(s)
Estructura Secundaria de Proteína , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/química , Algoritmos , Apoenzimas/biosíntesis , Apoenzimas/química , Apoenzimas/aislamiento & purificación , Sitios de Unión , Simulación por Computador , Escherichia coli/metabolismo , Hierro/metabolismo , Modelos Estructurales , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Superóxido Dismutasa/aislamiento & purificación , Tiorredoxinas/biosíntesis , Tiorredoxinas/metabolismoRESUMEN
The enzyme conferring resistance to the antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] originally reported by Suarez and co-workers [Area, P., Hardisson, C., & Suarez, J. E. (1990) Antimicrob. Agents Chemother. 34, 844-848] is demonstrated in this study to be a metalloglutathione transferase. The apoenzyme is a dimer of 16 kDa subunits. Electron paramagnetic resonance spectroscopy and water proton nuclear magnetic resonance longitudinal relaxation rates suggest that each subunit contains a mononuclear Mn2+ center that interacts strongly with the substrate fosfomycin (Kd = 17 microM) more weakly with the product (Kd = 1.1 mM) and very weakly or not at all with GSH. Inhomogeneous broadening of the EPR signals of enzyme-bound Mn2+ in the presence of H2(17)O indicates that three of the coordination sites on the metal are occupied by water. Sequence alignments, three-dimensional structures, and mechanistic considerations suggest that FosA is related to at least two other metalloenzymes, glyoxalase I and the Mn2+- or Fe2+-containing extradiol dioxygenases. The mechanistic imperative driving the evolution of this previously unidentified superfamily of metalloenzymes is proposed to be bidentate coordination of a substrate or intermediate to the metal center in the enzyme-catalyzed reactions.