Article

High temperatures enhance cooperative motions between CBM and catalytic domains of a thermostable cellulase: mechanism insights from essential dynamics

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
Physical Chemistry Chemical Physics (Impact Factor: 4.2). 06/2011; 13(30):13709-20. DOI: 10.1039/c0cp02697b
Source: PubMed

ABSTRACT Cellulases from thermophiles are capable of cleaving sugar chains from cellulose efficiently at high temperatures. The thermo-resistant Cel9A-68 cellulase possesses two important domains: CBM and a catalytic domain connected by a Pro/Ser/Thr rich linker. These domains act cooperatively to allow efficient catalysis. Despite exhaustive efforts to characterize cellulase binding and mechanism of action, a detailed description of the cellulose intrinsic flexibility is still lacking. From computational simulations we studied the temperature influence on the enzyme plasticity, prior to substrate binding. Interestingly, we observed an enhancement of collective motions at high temperatures. These motions are the most representative and describe an intrinsic hinge bending transition. A detailed analysis of these motions revealed an interdomain approximation where D459 and G460, located at the linker region, are the hinge residues. Therefore, we propose a new putative site for mutagenesis targeting the modulation of such conformational transition that may be crucial for activity.

Download full-text

Full-text

Available from: Paulo Ricardo Batista, Jul 15, 2015
0 Followers
 · 
152 Views
  • Source
    • "Cellulase enzymes belong to different families of glycoside hydrolases (GHs). Many cellulases contain a catalytic domain (CD) and a cellulose binding domain or module (Linder and Teeri, 1997); these are connected by a Pro/Ser/Thr rich linker (Batista et al., 2011). Other domains may also be present and these were recently described for bacterial cellulases in a comprehensive genome analysis (Medie et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM) to a synthetic glycosyl hydrolase improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the CBM of the tomato (Solanum lycopersicum) SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using carboxymethylcellulose, MUC, and native crystalline cellulose assays. The presence of the CBM substantially improved the endoglucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum bicolor plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.
    Frontiers in Plant Science 11/2012; 3:254. DOI:10.3389/fpls.2012.00254 · 3.95 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel β-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.
    Journal of molecular graphics & modelling 10/2012; 39C:29-38. DOI:10.1016/j.jmgm.2012.10.004 · 2.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Extremely thermophilic microorganisms have been sources of thermostable and thermoactive enzymes for over 30 years. However, information and insights gained from genome sequences, in conjunction with new tools for molecular genetics, have opened up exciting new possibilities for biotechnological opportunities based on extreme thermophiles that go beyond single-step biotransformations. Although the pace for discovering novel microorganisms has slowed over the past two decades, genome sequence data have provided clues to novel biomolecules and metabolic pathways, which can be mined for a range of new applications. Furthermore, recent advances in molecular genetics for extreme thermophiles have made metabolic engineering for high temperature applications a reality.
    11/2012; 1(4):363-372. DOI:10.1016/j.coche.2012.07.003
Show more