RESUMO
The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii beta-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank ) from T. reesei, and a beta-glucosidase from Aspergillus Niger (GenBank ), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported beta-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5 degrees C and half life of 62 min at 65 degrees C and was a specific beta-glucosidase with no beta-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (Vmax, 512 IU/mg) and was competitively inhibited by glucose (k(i), 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.
Assuntos
Regulação Enzimológica da Expressão Gênica , Talaromyces/enzimologia , Temperatura , Trichoderma/genética , beta-Glucosidase/química , beta-Glucosidase/genética , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática/fisiologia , Vetores Genéticos/genética , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Talaromyces/genética , Talaromyces/crescimento & desenvolvimento , beta-Glucosidase/isolamento & purificaçãoRESUMO
The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of approximately 60 A x 40 A x 50 A and an overall beta-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/genética , Genes Fúngicos , Talaromyces/enzimologia , Talaromyces/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Celulose 1,4-beta-Celobiosidase/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Glicosilação , Temperatura Alta , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Cellobiohydrolase IB is the first native enzyme from the filamentous fungus Talaromyces emersonii to be crystallized. It is a highly thermostable exo-acting enzyme. The native enzyme (MW = 56 kDa) was crystallized using the hanging-drop vapour-diffusion method with ammonium phosphate (dibasic) as a precipitant at pH 8.5. The crystal belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 74.43, c = 176.92 A, and diffracted to 1.77 A resolution at room temperature.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Talaromyces/química , Sítios de Ligação , Domínio Catalítico , Cristalização/métodos , Cristalografia por Raios X , Proteínas Fúngicas/química , Dados de Sequência Molecular , FosfatosRESUMO
The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures. Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.