Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei

VTT, Chemical Technology, Finaland.
Protein Science (Impact Factor: 2.86). 02/2008; 6(2):294-303. DOI: 10.1002/pro.5560060204
Source: PubMed

ABSTRACT Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.

Download full-text


Available from: Maija-Liisa Mattinen, Jul 28, 2015
  • Source
    • "The ThCel7A linker has two more potential O-glycosylation sites relative to TrCel7A. However, one of the extra threonines in ThCel7A (Thr 470) belongs to the CBM sequence and interacts with the core of the CBM in our molecular model build based on the experimental structure for T. reesei CBM (Mattinen et al. 1997). Therefore we chose to under-glycosylate the ThCel7A linker region rather than risking the addition of non-existing glycans in a site interacting with the CBM. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cellobiohydrolases hydrolyze cellulose releasing cellobiose units. They are very important for a number of biotechnological applications, such as, for example, production of cellulosic ethanol and cotton fiber processing. The Trichoderma cellobiohydrolase I (CBH1 or Cel7A) is an industrially important exocellulase. It exhibits a typical two domain architecture, with a small C-terminal cellulose-binding domain and a large N-terminal catalytic core domain, connected by an O-glycosylated linker peptide. The mechanism by which the linker mediates the concerted action of the two domains remains a conundrum. Here, we probe the protein shape and domain organization of the CBH1 of Trichoderma harzianum (ThCel7A) by small angle X-ray scattering (SAXS) and structural modeling. Our SAXS data shows that ThCel7A linker is partially-extended in solution. Structural modeling suggests that this linker conformation is stabilized by inter- and intra-molecular interactions involving the linker peptide and its O-glycosylations.
    Cellulose 08/2013; 20(4). DOI:10.1007/s10570-013-9933-3 · 3.03 Impact Factor
  • Source
    • "The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold are critical for specific binding with the aromatic rings stacked onto the glucose ring of the cellulose structure [14]. It is not certain at present how the lack of a CBM in the rScGE enzyme would influence its biochemical action on insoluble substrates. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 μM·min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.
    07/2012; 2012:951267. DOI:10.1155/2012/951267
  • Source
    • "The majority of the information concerning the role of CBDs was gathered by the use of domain exchange (Srisodsuk et al. 1997), domain removal (Van Tilbeurgh et al. 1986), or site-directed mutagenesis (Reinikainen et al. 1992, 1995, Mattinen et al. 1997). The fungus Trichoderma reesei produces several cellulases (Knowles et al. 1987), which show a similar structural organisation, i.e., a catalytic domain (or core domain), which is connected by a glycosylated linker to a core-binding domain (CBD) located at either the N or C terminal of the protein (Van Tilbeurgh et al. 1986, Abuja et al. 1988). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Core-binding domains of fungal cellulases from Trichoderma reesei were purified using a new and simple technique. Cellulases were hydrolysed with papain and the binding domains were then separated from the digested mixture by ultrafiltration. The enzymatic digestion process was monitored using capillary electrophoresis. This methodology produced a yield of 85% of binding domains.
    Biotechnology Letters 03/2000; 22(8):703-707. DOI:10.1023/A:1005691821173 · 1.74 Impact Factor
Show more