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1.
Anal Biochem ; 471: 44-50, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25447496

RESUMEN

Ethylene is a key molecule in organic synthesis currently produced by steam cracking of fossil hydrocarbons. In nature, ethylene is produced in higher plants by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). Biocatalytic alternatives for ethylene production are still far from being competitive with traditional production plants. Furthermore, data dispersion shown in the literature adds uncertainty to the introduction of ACCO as a biocatalyst, especially when larger numbers of isoforms or mutants are to be compared. Here we propose a new method for measuring ACCO activity based on cyanide detection. Data provided here indicate that cyanide detection is more precise, more responsive, and much more stable than any other method tested for ACCO activity estimation so far. Briefly, enzymatically produced cyanide can be detected by its derivatization with naphthalene-2,3-dicarboxyaldehide (NDA) to generate 1-cyanobenz[f]isoindole (CBI), which is further detected by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector. Cyanide can be detected in the range between 0.99 and 60.17pmol, which is three orders of magnitude more sensitive than the currently used ethylene estimation method.


Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Pruebas de Enzimas/métodos , Nitrilos/metabolismo , Indoles/química , Solanum lycopersicum/enzimología , Nitrilos/química
2.
Microb Cell Fact ; 13(1): 25, 2014 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-24533452

RESUMEN

BACKGROUND: Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein. RESULTS: The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased. CONCLUSIONS: Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of oxidation in protein assembly is studied, evidencing that oxidation should be minimized during the production process if VP6 nanotubes are required.


Asunto(s)
Antígenos Virales/química , Proteínas de la Cápside/química , Metales/química , Rotavirus/fisiología , Animales , Antígenos Virales/genética , Antígenos Virales/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Bovinos , Compuestos Ferrosos/química , Peróxido de Hidrógeno/química , Luz , Nanotubos/química , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Dispersión de Radiación , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Ensamble de Virus
3.
BMC Microbiol ; 12: 249, 2012 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-23122209

RESUMEN

BACKGROUND: Different systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components. RESULTS: By the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires. CONCLUSIONS: These observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems.


Asunto(s)
Cobre/metabolismo , Evolución Molecular , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Variación Genética , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Biología Computacional , Genoma Bacteriano , Homeostasis , Filogenia
4.
PLoS One ; 7(7): e40700, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22808237

RESUMEN

Molecular dynamic (MD) simulations have been performed on Tth-MCO, a hyperthermophilic multicopper oxidase from thermus thermophilus HB27, in the apo as well as the holo form, with the aim of exploring the structural dynamic properties common to the two conformational states. According to structural comparison between this enzyme and other MCOs, the substrate in process to electron transfer in an outer-sphere event seems to transiently occupy a shallow and overall hydrophobic cavity near the Cu type 1 (T1Cu). The linker connecting the ß-strands 21 and 24 of the second domain (loop (ß21-ß24)(D2)) has the same conformation in both states, forming a flexible lid at the entrance of the electron-transfer cavity. Loop (ß21-ß24)(D2) has been tentatively assigned a role occluding the access to the electron-transfer site. The dynamic of the loop (ß21-ß24)(D2) has been investigated by MD simulation, and results show that the structures of both species have the same secondary and tertiary structure during almost all the MD simulations. In the simulation, loop (ß21-ß24)(D2) of the holo form undergoes a higher mobility than in the apo form. In fact, loop (ß21-ß24)(D2) of the holo form experiences a conformational change which enables exposure to the electron-transfer site (open conformation), while in the apo form the opposite effect takes place (closed conformation). To confirm the hypothesis that the open conformation might facilitate the transient electron-donor molecule occupation of the site, the simulation was extended another 40 ns with the electron-donor molecule docked into the protein cavity. Upon electron-donor molecule stabilization, loops near the cavity reduce their mobility. These findings show that coordination between the copper and the protein might play an important role in the general mobility of the enzyme, and that the open conformation seems to be required for the electron transfer process to T1Cu.


Asunto(s)
Apoenzimas/química , Cobre/metabolismo , Holoenzimas/química , Simulación de Dinámica Molecular , Oxidorreductasas/química , Thermus thermophilus/enzimología , Cristalografía por Rayos X , Estabilidad de Enzimas , Oxidorreductasas/metabolismo , Estructura Secundaria de Proteína , Temperatura
5.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 5): 564-77, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22525754

RESUMEN

X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV-Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O(2) reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account.


Asunto(s)
Basidiomycota/enzimología , Dominio Catalítico/efectos de la radiación , Cobre/química , Cristalografía por Rayos X , Lacasa/química , Cristalización , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Oxidación-Reducción , Oxígeno/química , Conformación Proteica/efectos de la radiación , Espectrofotometría Ultravioleta , Rayos X
6.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 12): 1595-8, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22139175

RESUMEN

A thermostable multicopper oxidase from Thermus thermophilus HB27 (Tth-MCO) was successfully crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. Crystallization conditions and preliminary X-ray diffraction data to 1.5 Å resolution obtained using synchrotron radiation at 100 K are reported. The crystals belonged to space group C222(1), with unit-cell parameters a = 93.6, b = 110.3, c = 96.3 Å. A monomer in the asymmetric unit yielded a Matthews coefficient (V(M)) of 2.60 Å(3) Da(-1) and a solvent content of 53%. An inactive enzyme form, apo-Tth-MCO, was also crystallized and diffraction data were collected to 1.7 Å resolution. In addition, a second inactive form of the enzyme, Hg-Tth-MCO, was obtained by soaking apo-Tth-MCO crystals with mercury(II) chloride and data were collected to a resolution of 1.7 Å.


Asunto(s)
Oxidorreductasas/química , Thermus thermophilus/enzimología , Apoenzimas/química , Cristalografía por Rayos X , Estabilidad de Enzimas , Holoenzimas/química
7.
Antioxid Redox Signal ; 11(2): 167-92, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19099403

RESUMEN

Redox-active enzymes perform many key biological reactions. The electron transfer process is complex, not only because of its versatility, but also because of the intricate and delicate modulation exerted by the protein scaffold on the redox properties of the catalytic sites. Nowadays, there is a wealth of information available about the catalytic mechanisms of redox-active enzymes and the time is propitious for the development of projects based on the protein engineering of redox-active enzymes. In this review, we aim to provide an updated account of the available methods used for protein engineering, including both genetic and chemical tools, which are usually reviewed separately. Specific applications to redox-active enzymes are mentioned within each technology, with emphasis on those cases where the generation of novel functionality was pursued. Finally, we focus on two emerging fields in the protein engineering of redox-active enzymes: the construction of novel nucleic acid-based catalysts and the remodeling of intra-molecular electron transfer networks. We consider that the future development of these areas will represent fine examples of the concurrence of chemical and genetic tools.


Asunto(s)
Enzimas/química , Enzimas/metabolismo , Ingeniería de Proteínas/métodos , Enzimas/síntesis química , Oxidación-Reducción , Transducción de Señal
8.
Microbiol Immunol ; 52(10): 492-8, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18822083

RESUMEN

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.


Asunto(s)
Cryptococcus neoformans/enzimología , Lacasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cryptococcus neoformans/genética , ADN de Hongos/química , ADN de Hongos/genética , Variación Genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN
9.
FASEB J ; 20(8): 1233-5, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16720736

RESUMEN

Iso-1-cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox-inspired strategy was implemented until an iso-1-cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15-fold higher total-turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein-based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox-inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.


Asunto(s)
Citocromos c/química , Citocromos c/genética , Ingeniería de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Catálisis , Citocromos c/metabolismo , Peróxido de Hidrógeno/química , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
J Biotechnol ; 117(1): 73-82, 2005 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-15831249

RESUMEN

The incorporation of fungal laccases into novel applications has been delayed mainly due to their intrinsic sensitivity towards halides and alkaline conditions. In order to explore new sources of enzymes we evaluated the multipotent polyphenol oxidase PPO1 from the marine bacterium Marinomonas mediterranea. Here we report that, in contrast to its fungal counterparts, PPO1 remained functional above neutral pH presenting high specificity for phenolic compounds, in particular for methoxyl-substituted mono-phenols and catechols. These properties, in addition to its tolerance towards chloride (up to 1 M) and its elevated redox potential at neutral pH (0.9 V), suggest this enzyme may be an interesting candidate for specific applications such as the Amperometric determination of phenolic compounds and bio-fuel cells.


Asunto(s)
Catecol Oxidasa/metabolismo , Gammaproteobacteria/enzimología , Catálisis , Catecol Oxidasa/química , Cloruros/farmacología , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Especificidad por Sustrato
11.
Biochemistry ; 44(11): 4267-74, 2005 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-15766255

RESUMEN

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.


Asunto(s)
Basidiomycota/enzimología , Dominio Catalítico , Peroxidasas/química , Triptófano/química , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Radicales Libres/química , Modelos Moleculares , Peroxidasas/aislamiento & purificación , Peroxidasas/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato
12.
FEMS Microbiol Lett ; 244(2): 235-41, 2005 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-15766774

RESUMEN

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.


Asunto(s)
Basidiomycota/enzimología , Lacasa/clasificación , Secuencia de Aminoácidos , Basidiomycota/genética , Biodegradación Ambiental , Lacasa/biosíntesis , Lacasa/química , Lacasa/genética , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido
13.
Biodegradation ; 14(6): 397-406, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14669870

RESUMEN

The enzymatic mechanism for the transformation of organophosphorus pesticides (OPPs) by different white-rot fungi strains was studied. With the exception of Ganoderma applanatum 8168, all strains from a collection of 17 different fungi cultures were able to deplete parathion. Three strains showing the highest activities were selected for further studies: Bjerkandera adusta 8258, Pleurotus ostreatus 7989 and Phanerochaete chrysosporium 3641. These strains depleted 50 to 96% of terbufos, azinphos-methyl, phosmet and tribufos after four-days exposure to the pesticides. In order to identify the cellular localization of the transformation activity, the extracellular and microsomal fractions of Pleuronts ostreatus 7989 were evaluated in vitro. While the activities of ligninolytic enzymes (lignin peroxidase, manganese peroxidase and laccase) were detected in the extracellular fraction, no enzymatic modification of any of the five pesticides tested could be found, suggesting the intracellular origin of the transformation activity. In accordance with this observation the microsomal fraction was found able to transform three OPPs with the following rates: 10 micromol mg prot(-1) h(-1) for phosmet, 5.7 micromol mg prot(-1) h(-1) for terbufos, and 2.2 micromol mg prot(-1) h(-1) for azinphos-methyl. The products from these reactions and from the transformation of trichlorfon and malathion, were identified by mass-spectrometry. These results, supported by specific inhibition experiments and the stringent requirement for NADPH during the in vitro assays suggest the involvement of a cytochrome P450.


Asunto(s)
Hongos/metabolismo , Microsomas/metabolismo , Plaguicidas/metabolismo , Biodegradación Ambiental , Biotransformación , Inhibidores de la Colinesterasa/metabolismo , Enzimas/metabolismo , Proteínas Fúngicas/metabolismo , Hongos/enzimología , Hongos/ultraestructura , Microsomas/enzimología , Compuestos Organofosforados/metabolismo
14.
Antonie Van Leeuwenhoek ; 84(4): 289-99, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-14574106

RESUMEN

Fungal laccases have been extensively exploited for industrial purposes and there is a wealth of information available regarding their reaction mechanism, biological role and several molecular aspects, including cloning, heterologous expression and transcriptional analyses. Here we present the reconstruction of the fungal laccase loci evolution inferred from the comparative analysis of 48 different sequences. The topology of the phylogenetic trees indicate that a single monophyletic branch exists for fungal laccases and that laccase isozyme genes may have evolved independently, possibly through duplication-divergence events. Laccases are copper-containing enzymes generally identified by the utilization of substituted p-diphenol substrates. Interestingly, our approach permitted the assignment of two copper-containing oxidases, preliminarily catalogued as laccases, to a different evolutionary group, distantly related to the main branch of bona fide laccases.


Asunto(s)
Evolución Molecular , Hongos/enzimología , Hongos/genética , Lacasa/química , Lacasa/genética , Sitios de Unión , Cobre/metabolismo , Hongos/clasificación , Duplicación de Gen , Datos de Secuencia Molecular , Estructura Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido
15.
J Bacteriol ; 185(20): 5976-83, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14526008

RESUMEN

Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits. We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer. Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter. RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P. aeruginosa virulence-associated traits.


Asunto(s)
4-Butirolactona/análogos & derivados , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hexosiltransferasas/metabolismo , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/crecimiento & desarrollo , Transcripción Genética , 4-Butirolactona/metabolismo , Proteínas Bacterianas/genética , Medios de Cultivo , Escherichia coli/genética , Escherichia coli/metabolismo , Hexosiltransferasas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transducción de Señal
16.
FEBS Lett ; 551(1-3): 63-70, 2003 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-12965206

RESUMEN

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) is an allosteric enzyme that catalyzes the reversible conversion of D-glucosamine-6-phosphate into D-fructose-6-phosphate and ammonium. Here we describe the existence of two mammalian glucosamine-6-phosphate deaminase enzymes. We present the crystallographic structure of one of them, the long human glucosamine-6-phosphate deaminase, at 1.75 A resolution. Crystals belong to the space group P2(1)2(1)2(1) and present a whole hexamer in the asymmetric unit. The active-site lid (residues 162-182) presented significant structural differences among monomers. Interestingly the region with the largest differences, when compared with the Escherichia coli homologue, was found to be close to the active site. These structural differences can be related to the kinetic and allosteric properties of both mammalian enzymes.


Asunto(s)
Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/genética , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cricetinae , Cristalografía por Rayos X , Escherichia coli/enzimología , Humanos , Isoenzimas/química , Isoenzimas/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
17.
Bioconjug Chem ; 13(6): 1336-44, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12440871

RESUMEN

Chemically modified cytochrome c with poly(ethylene glycol) (PEG) showed activity at temperatures higher than 100 degrees C and to be highly thermostable. The molecular size of PEG moieties and the coupling site affected the thermal stabilization. An optimal PEG/protein mass ratio of 2.8 was found, producing a fully thermostable biocatalyst at 80 degrees C. Site-directed mutagenesis on yeast cytochrome c showed an increased thermostabilization when lysine 79 residue, localized at the edge of the active site, was replaced by a nonreactive residue. Tertiary, secondary, and active-site structures were analyzed by fluorescence, CD, and UV/visible spectroscopies. Besides its disordered structure, the pegylated protein showed a lower unfolding rate at the active-site than the unmodified ones. A shell-like structure seems to protect the heme environment, in which PEG is coiled on the protein surface with a primary shield of rigid water molecules solvating the hydrophilic region of bound-PEG, and the PEG hydrophobic regions interacting with the hydrophobic clusters on protein surface.


Asunto(s)
Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Calor , Animales , Sitios de Unión , Catálisis , Grupo Citocromo c/genética , Caballos , Miocardio , Oxidación-Reducción , Compuestos Policíclicos/química , Polietilenglicoles/química , Pliegue de Proteína , Saccharomyces cerevisiae , Análisis Espectral , Termodinámica
18.
Chem Biol ; 9(5): 555-65, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-12031662

RESUMEN

As the number of industrial applications for proteins continues to expand, the exploitation of protein engineering becomes critical. It is predicted that protein engineering can generate enzymes with new catalytic properties and create desirable, high-value, products at lower production costs. Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications. However, peroxidases are unstable and are readily inactivated by their substrate, hydrogen peroxide. Researchers rely on the powerful tools of molecular biology to improve the stability of these enzymes, either by protecting residues sensitive to oxidation or by devising more efficient intramolecular pathways for free-radical allocation. Here, we discuss the catalytic cycle of peroxidases and the mechanism of the suicide inactivation process to establish a broad knowledge base for future rational protein engineering.


Asunto(s)
Peróxido de Hidrógeno/metabolismo , Peroxidasas/antagonistas & inhibidores , Peroxidasas/metabolismo , Ingeniería de Proteínas/métodos , Catálisis , Bases de Datos de Proteínas , Estabilidad de Enzimas , Radicales Libres/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Peroxidasas/química , Conformación Proteica
19.
Microbiology (Reading) ; 146 ( Pt 7): 1627-1637, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10878127

RESUMEN

The isolation and characterization of a Rhizobium etli glutamate auxotroph, TAD12, harbouring a single Tn5 insertion, is reported. This mutant produced no detectable glutamate synthase (GOGAT) activity. The cloning and physical characterization of a 7.2 kb fragment of R. etli DNA harbouring the structural genes gltB and gltD encoding the two GOGAT subunits GltB and GltD is also reported. In comparison with the wild-type strain (CFN42), the GOGAT mutant strain utilized less succinate and glutamate and grew less with this and other amino acids as nitrogen source. R. etli assimilates ammonium by the glutamine synthetase (GS)-GOGAT pathway and a GOGAT mutant prevents the cycling of glutamine by this pathway, something that impairs nitrogen and carbon metabolism and explains the decrease in the amino-nitrogen during exponential growth, with glutamate as nitrogen source. GOGAT activity also has a role in ammonium turnover and in the synthesis of amino acids and proteins, processes that are necessary to sustain cell viability in non-growing conditions. The assimilation of ammonium is important during symbiosis and glutamate constitutes 20-40% of the total amino-nitrogen. In symbiosis, the blockage of ammonium assimilation by a GOGAT mutation significantly decreases the amino-nitrogen pool of the bacteroids and may explain why more N(2) is fixed in ammonium, excreted to the plant cell, transported to the leaves and stored in the seeds.


Asunto(s)
Carbono/metabolismo , Glutamato Sintasa/genética , Nitrógeno/metabolismo , Rhizobium/genética , Secuencia de Aminoácidos , Glutamato Sintasa/metabolismo , Ácido Glutámico/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Compuestos de Amonio Cuaternario/metabolismo , Rhizobium/enzimología , Rhizobium/crecimiento & desarrollo , Alineación de Secuencia , Ácido Succínico/metabolismo
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