PDBsum entry 1q2b

Hydrolase

PDB id: 1q2b

Contents

Protein chain

434 a.a. *

Ligands

NAG

Metals

_CO ×2

Waters ×357

* Residue conservation analysis

PDB id:

1q2b

Name:

Hydrolase

Title:

Cellobiohydrolase cel7a with disulphide bridge added across exo-loop by mutations d241c and d249c

Structure:

Exocellobiohydrolase i. Chain: a. Fragment: catalytic domain 1-434. Synonym: exoglucanase i. Cbhi. 1,4-beta-cellobiohydrolase. Cellulose 1,4-beta-cellobiosidase. Engineered: yes. Mutation: yes

Source:

Hypocrea jecorina. Organism_taxid: 51453. Strain: qm9414. Variant: vtt-d-93201. Gene: cbh1. Expressed in: hypocrea jecorina. Expression_system_taxid: 51453.

Resolution:

1.60Å R-factor: 0.207 R-free: 0.223

Authors:

J.Stahlberg,M.Harris,T.A.Jones

Key ref:

I.von Ossowski et al. (2003). Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D. J Mol Biol, 333, 817-829. PubMed id: 14568538 DOI: 10.1016/S0022-2836(03)00881-7

Date:

24-Jul-03 Release date: 25-Nov-03

Protein chain

P62694  (GUX1_HYPJE) -  Exoglucanase 1 from Hypocrea jecorina

Seq: 513 a.a.

Struc: 434 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.91 - cellulose 1,4-beta-cellobiosidase (non-reducing end).
• Reaction: Hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains.

DOI no: 10.1016/S0022-2836(03)00881-7

J Mol Biol 333:817-829 (2003)

PubMed id: 14568538

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D.

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.

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.

Selected figure(s)

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.

Figures were selected by an automated process.