RESUMO
Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.
Assuntos
Celulase/química , Celulase/fisiologia , Proteínas Fúngicas/fisiologia , Piromyces/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Celulase/genética , Celulose 1,4-beta-Celobiosidase , Clonagem Molecular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Biblioteca Gênica , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/farmacologiaRESUMO
Since the discovery of naturally occurring non-protein enzymes called ribozymes, which cleave RNAs with high specificity akin to biological re-usable scissors, much progress has been made in understanding their mechanisms of action, improving their stability and effectiveness, and in investigating their clinical usage. Owing to their high specificity, broad range of target selection and action prior to protein translation, ribozymes are considered to be an ideal approach to gene therapy. However, the lack of suitable delivery systems to deliver ribozymes to target cells hampers their clinical development.
