RESUMEN

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.

Asunto(s)

Proteínas Bacterianas/metabolismo , Celulosa/metabolismo , Clostridium/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/genética , Biotecnología/métodos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Celulasa/genética , Celulasa/metabolismo , Clostridium/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genética

RESUMEN

Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the beta-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-A resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family III(c) cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 A, respectively.

Asunto(s)

Celulasa/química , Celulosa/metabolismo , Clostridium/enzimología , Oligosacáridos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Celobiosa/metabolismo , Celulasa/metabolismo , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia

RESUMEN

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.

Asunto(s)

Proteínas Bacterianas/química , Dominio Catalítico , Celulasa/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Calcio/química , Clostridium/enzimología , Cristalización , Cristalografía por Rayos X , Glicósido Hidrolasas/química , Datos de Secuencia Molecular , Níquel/química , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Zinc/química

RESUMEN

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

Asunto(s)

Celulasa/metabolismo , Celulosa/metabolismo , Clostridium/genética , Clostridium/metabolismo , Secuencias de Aminoácidos , Secuencia de Bases , Sitios de Unión , Celulasa/genética , Cartilla de ADN , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellum and Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of the C. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (K(a) approximately 10(9) m(-1)) by a cohesin from C. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.

Asunto(s)

Celulasa/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencias de Aminoácidos , Biotinilación , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Clostridium/química , Clostridium/metabolismo , ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas , Vectores Genéticos , Hidroxilación , Cinética , Leucina/química , Ligandos , Mutagénesis Sitio-Dirigida , Mutación , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Programas Informáticos , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Treonina/química , Cohesinas

RESUMEN

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV x cm(-1) field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV x cm(-1) pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10(5) and 10(7) transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively.

Asunto(s)

Clostridium/genética , Transformación Bacteriana/genética , Permeabilidad de la Membrana Celular , Electricidad , Plásmidos , Selección Genética

RESUMEN

The crystal structure of the family IIIa cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit (CipC) of Clostridium cellulolyticum has been determined. The structure reveals a nine-stranded jelly-roll topology which exhibits distinctive structural elements consistent with family III CBDs that bind crystalline cellulose. These include a well conserved calcium-binding site, a putative cellulose-binding surface and a conserved shallow groove of unknown function. The CipC CBD structure is very similar to the previously elucidated family IIIa CBD from the CipA scaffoldin of C. thermocellum, with some minor differences. The CipC CBD structure was also compared with other previously described CBD structures from families IIIc and IV derived from the endoglucanases of Thermomonospora fusca and Cellulomonas fimi, respectively. The possible functional consequences of structural similarities and differences in the shallow groove and cellulose-binding faces among various CBD families and subfamilies are discussed.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Portadoras/química , Clostridium/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Celulosa/metabolismo , Cristalografía por Rayos X , Proteínas de la Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido

RESUMEN

In the assembly of the Clostridium cellulolyticum cellulosome, the multiple cohesin modules of the scaffolding protein CipC serve as receptors for cellulolytic enzymes which bear a dockerin module. The X-ray structure of a type I C. cellulolyticum cohesin module (Cc-cohesin) has been solved using molecular replacement, and refined at 2.0 A resolution. Despite a rather low sequence identity of 32 %, this module has a fold close to those of the two Clostridium thermocellum cohesin (Ct-cohesin) modules whose 3D structures have been determined previously. Cc-cohesin forms a dimer in the crystal, as do the two Ct-cohesins. We show here that the dimer exists in solution and that addition of dockerin-containing proteins dissociates the dimer. This suggests that the dimerization interface and the cohesin/dockerin interface may overlap. The nature of the overall surface and of the dimer interface of Cc-cohesin differ notably from those of the Ct-cohesin modules, being much less polar, and this may explain the species specificity observed in the cohesin/dockerin interaction of C. cellulolyticum and C. thermocellum. We have produced a topology model of a C. cellulolyticum dockerin and of a Cc-cohesin/dockerin complex using homology modeling and available biochemical data. Our model suggests that a special residue pair, already identified in dockerin sequences, is located at the center of the cohesin surface putatively interacting with the dockerin.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Clostridium/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía en Gel , Cristalografía por Rayos X , Dimerización , Proteínas de la Membrana/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad de la Especie , Especificidad por Sustrato

RESUMEN

Multidimensional, homo- and heteronuclear magnetic resonance spectroscopy combined with dynamical annealing has been used to determine the structure of a 94 residue module (X2 1) of the scaffolding protein CipC from the anaerobic bacterium Clostridium cellulolyticum. An experimental data set comprising 1647 nuclear Overhauser effect-derived restraints, 105 hydrogen bond restraints and 66 phi torsion angle restraints was used to calculate 20 converging final solutions. The calculated structures have an average rmsd about the mean structure of 0.55(+/-0.11) A for backbone atoms and 1.40(+/-0.11) A for all heavy atoms when fitted over the secondary structural elements. The X2 1 module has an immunoglobulin-like fold with two beta-sheets packed against each other. One sheet contains three strands, the second contains four strands. An additional strand is intercalated between the beta-sandwich, as well as two turns of a 3(.10) helix. X2 1 has a surprising conformational stability and may act as a conformational linker and solubility enhancer within the scaffolding protein. The fold of X2 1 is very similar to that of telokin, titin Ig domain, hemolin D2 domain, twitchin immunoglobulin domain and the first four domains of the IgSF portion of transmembrane cell adhesion molecule. As a consequence, the X2 1 module is the first prokaryotic member assigned to the I set of the immunoglobulin superfamily even though no sequence similarity with any member of this superfamily could be detected.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Portadoras/química , Clostridium/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Estructuras Citoplasmáticas/química , Enlace de Hidrógeno , Inmunoglobulinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Electricidad Estática

RESUMEN

Cellulase Cel48F from Clostridium cellulolyticum was described as a processive endo-cellulase. The active site is composed of a 25 A long tunnel which is followed by an open cleft. During the processive action, the cellulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage. To study this processive action in the tunnel, the native catalytic module of Cel48F and the inactive mutant E55Q, have been cocrystallized with cellobiitol, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oligosaccharides cellobiose, -tetraose and -hexaose. Seven sub-sites in the tunnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed. The sub-sites observed for the thio-oligosaccharide inhibitors and oligosaccharides, respectively, were located at two different positions in the tunnel corresponding to a shift in the chain direction of about a half sugar subunit. These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel. Multiple patterns are not observed in nonprocessive endo-cellulases, where only one sugar position is favored by aromatic stacking. It is therefore proposed that the aromatic residues serve as lubricating agents to reduce the sliding barrier in the processive action.

Asunto(s)

Celulasa/antagonistas & inhibidores , Celulasa/química , Celulosa/análogos & derivados , Inhibidores Enzimáticos/química , Sitios de Unión/genética , Celobiosa/química , Celulasa/genética , Celulosa/química , Clostridium/enzimología , Clostridium/genética , Cristalografía por Rayos X , Enlace de Hidrógeno , Hidrólisis , Sustancias Macromoleculares , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligosacáridos/química , Especificidad por Sustrato/genética , Tetrosas/química

RESUMEN

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the approximately 70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170-177.

Asunto(s)

Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Proteínas de la Membrana/metabolismo , Marcadores de Afinidad , Sustitución de Aminoácidos , Bacillus/química , Proteínas Bacterianas/química , Sitios de Unión , Celulasa/química , Celulasa/genética , Clostridium/química , Proteínas de la Membrana/química , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie

RESUMEN

CelE, one of the three major proteins of the cellulosome of Clostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on p-nitrophenyl-cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.

Asunto(s)

Proteínas Bacterianas , Celulasa/química , Celulasa/metabolismo , Clostridium/enzimología , Orgánulos/enzimología , Sitios de Unión , Catálisis , Celobiosa/análogos & derivados , Celobiosa/metabolismo , Celulasa/genética , Celulasa/aislamiento & purificación , Celulosa/análogos & derivados , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa , Clonación Molecular , Clostridium/citología , Cristalización , Sinergismo Farmacológico , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Solubilidad , Especificidad por Sustrato , Termodinámica , Viscosidad

RESUMEN

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Clostridium/enzimología , Complejos Multienzimáticos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Calcio/metabolismo , Proteínas Portadoras/química , Celulosa/metabolismo , Dicroismo Circular , Cartilla de ADN , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279-2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013-1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 x 10(8) M-1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clostridium/genética , Clostridium/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Celulasa/metabolismo , Cartilla de ADN/genética , ADN Bacteriano/genética , Péptidos y Proteínas de Señalización Intracelular , Cinética , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Sistemas de Lectura Abierta , Filogenia , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo

RESUMEN

The catalytic domain of the CeIF processive endocellulase, a family 48 glycosyl hydrolase from Clostridium cellulolyticum has been crystallized in the presence of a newly synthesized inhibitor (methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside), by vapour diffusion, using PEG as a precipitant. The protein crystallizes in the orthorhombic P212121 space group and diffracts to a resolution of 2.0 A. The unit-cell parameters are a = 61.4, b = 84.5, c = 121.9 A.

Asunto(s)

Celulasa/análisis , Celulasa/química , Clostridium/enzimología , Inhibidores Enzimáticos/farmacología , Oligosacáridos/química , Secuencia de Carbohidratos , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Molecular

RESUMEN

The mesophilic bacterium Clostridium cellulolyticum exports multienzyme complexes called cellulosomes to digest cellulose. One of the three major components of the cellulosome is the processive endocellulase CelF. The crystal structure of the catalytic domain of CelF in complex with two molecules of a thiooligosaccharide inhibitor was determined at 2.0 A resolution. This is the first three-dimensional structure to be solved of a member of the family 48 glycosyl hydrolases. The structure consists of an (alpha alpha)6-helix barrel with long loops on the N-terminal side of the inner helices, which form a tunnel, and an open cleft region covering one side of the barrel. One inhibitor molecule is enclosed in the tunnel, the other exposed in the open cleft. The active centre is located in a depression at the junction of the cleft and tunnel regions. Glu55 is the proposed proton donor in the cleavage reaction, while the corresponding base is proposed to be either Glu44 or Asp230. The orientation of the reducing ends of the inhibitor molecules together with the chain translation through the tunnel in the direction of the active centre indicates that CelF cleaves processively cellobiose from the reducing to the non-reducing end of the cellulose chain.

Asunto(s)

Celobiosa/análogos & derivados , Celulasa/química , Clostridium/enzimología , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Dominio Catalítico , Celobiosa/química , Celulasa/antagonistas & inhibidores , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Estructura Secundaria de Proteína

RESUMEN

The ability of Desulfovibrio fructosovorans MR400 DeltahynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon.

Asunto(s)

Desulfovibrio/enzimología , Hidrogenasas/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Desulfovibrio/genética , Espectroscopía de Resonancia por Spin del Electrón , Hidrogenasas/química , Datos de Secuencia Molecular , Operón

RESUMEN

The NADP-reducing hydrogenase of Desulfovibrio fructosovorans represents a novel class of [Fe] hydrogenases which is encoded by the well-characterized hndABCD operon containing the genes hndA, hndB, hndC, and hndD. Expression of this operon, monitored by measuring the NADP-reducing activity, was found to be maximum during the exponential phase of growth on fructose and then decreased when the concentration of the carbon and energy source became limiting. The optimum pH for the H2-driven NADP reduction was 8, and the apparent K(m) and Vmax were determined to be 0.09 mM and 13 x 10(-3) u/mg, respectively. Heterologous expression of the hnd genes in Escherichia coli was carried out to raise antisera against the different subunits of the NADP-reducing hydrogenase. The antisera were used to detect the four subunits in cell extract of D. fructosovorans after separation by SDS- and native PAGE. The four subunits of the NADP-reducing hydrogenase were demonstrated to be associated in a complex which exhibited H2-driven methyl viologen reduction. Furthermore, on native gel, a form lacking HndD, with no hydrogen-dependent methyl viologen reductase activity was also shown to be present in D. fructosovorans.

Asunto(s)

Desulfovibrio/enzimología , Oxidorreductasas/metabolismo , Proteínas Bacterianas , Cartilla de ADN , Desulfovibrio/genética , Desulfovibrio/crecimiento & desarrollo , Amplificación de Genes , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Oxidorreductasas/biosíntesis , Oxidorreductasas/química , Paraquat/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.

Asunto(s)

Desulfovibrio/enzimología , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Bacterianas , Secuencia de Bases , Clostridium/enzimología , Clostridium/genética , Cartilla de ADN/genética , Desulfovibrio/genética , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Ferredoxinas/química , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/aislamiento & purificación , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/genética , Paracoccus denitrificans/enzimología , Paracoccus denitrificans/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido

RESUMEN

The pBG1 replicon from the cryptic plasmid of Desulfovibrio desulfuricans G100A was inserted into pTZ18U derivatives to generate a new family of shuttle vectors. These plasmids are stable both in Escherichia coli and in Desulfovibrio, they present a large number of unique restriction sites, and colonies of recombinant clones can be identified by blue/white screening in E. coli. The pBMC, pBMK, and pBMS series carry the cat, npt, or strAB genes as selectable markers, respectively. The pBMC6, pBMK6, and pBMS6 plasmids can be introduced both in D. desulfuricans and in Desulfovibrio fructosovorans by electrotransformation, and the pBMC7, pBMK7, and pBMS7 plasmids contain additional mobilization functions which makes them suitable for conjugation.

Asunto(s)

Clonación Molecular/métodos , Desulfovibrio/genética , Vectores Genéticos/genética , Conjugación Genética , Electroporación , Escherichia coli/genética , Hidrogenasas/genética , Plásmidos/genética , Replicón/genética , Transformación Bacteriana