PDBsum entry 1q2b

Hydrolase

PDB id: 1q2b

Contents

Protein chain

434 a.a. *

Ligands

NAG

Metals

_CO ×2

Waters ×357

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Engineering the exo-Loop of trichoderma reesei cellobiohydrolase, Cel7a. A comparison with phanerochaete chrysosporium cel7d.

Authors

I.Von ossowski, J.Ståhlberg, A.Koivula, K.Piens, D.Becker, H.Boer, R.Harle, M.Harris, C.Divne, S.Mahdi, Y.Zhao, H.Driguez, M.Claeyssens, M.L.Sinnott, T.T.Teeri.

Ref.

J Mol Biol, 2003, 333, 817-829. [DOI no: 10.1016/S0022-2836(03)00881-7]

PubMed id

14568538

Abstract

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

Figure 3.
Figure 3. Comparison of the structure of the exo-loop in the D241C/D249C disulphide mutant (pink) and in the complex of Cel7A E217Q (wheat) with cellohexaose and cellobiose (PDB entry 7CEL), together with a model of a cellulose chain from PDB entry 8CEL.[4.] Hydrogen bonds in the 7CEL structure are indicated with small white spheres.

Figure 4.
Figure 4. Comparison of (a) temperature factors in the exo-loop region, and (b) C^a-distances between the structures of the D241C/D249C disulphide mutant and the unliganded Cel7A wild-type (APO) or the E217Q-cellohexaose complex (7CEL),[4.] respectively.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 333, 817-829) copyright 2003.

Secondary reference #1

Title

Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase i from trichoderma reesei.

Authors

J.Ståhlberg, C.Divne, A.Koivula, K.Piens, M.Claeyssens, T.T.Teeri, T.A.Jones.

Ref.

J Mol Biol, 1996, 264, 337-349. [DOI no: 10.1006/jmbi.1996.0644]

PubMed id

8951380

Figure 2.
Figure 2. Close-up view of a superposition of the CBHI wild-type and mutant active sites: wild-type/IBTG (beige), E212Q (blue), D214N (magenta) and E212Q/cel- lobiose (green). Only the residues close to the cleavage site are shown. For clarity, the ligands and water molecules have been omitted. The residue types given refer to those of wild-type CBHI. In the D214N model, a calcium ion is bound to Glu212. The side-chain of Gln175 flips to participate in metal co-ordination. The illustration was created using the program O (Jones et al., 1991).

Figure 5.
Figure 5. Superposition of residues in the active site of CBHI (beige) and the Bacillus macerans 1,3-1,4-b-glu- canase (blue; PDB accession code 1MAC). The side-chains are presented as ball-and-stick models.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

High-Resolution crystal structures reveal how a cellulose chain is bound in the 50 a long tunnel of cellobiohydrolase i from trichoderma reesei.

Authors

C.Divne, J.Ståhlberg, T.T.Teeri, T.A.Jones.

Ref.

J Mol Biol, 1998, 275, 309-325. [DOI no: 10.1006/jmbi.1997.1437]

PubMed id

9466911

Figure 1.
Figure 1. Schematic representation of the CBHI catalytic domain with a cellooligomer bound in sites −7 to +2. Secondary-structure elements are coloured as follows: β strands, blue arrows; α helices, red spirals; loop regions, yellow coils. The cellooligomer is shown in pink as a ball-and-stick object. The illustration was created with MOLSCRIPT (Kraulis, 1991).

Figure 5.
Figure 5. Tryptophan residues from T. reesei CBHI (yellow) and CBHII (red) aligned with respect to a single glucose residue. Tryptophan-indole rings interacting with the more hydrophobic β face a shown “above” the glucosyl unit, and those interacting with the α face “below”.

The above figures are reproduced from the cited reference with permission from Elsevier