PDBsum entry 4cel

Hydrolase

PDB id: 4cel

Contents

Protein chains

434 a.a. *

Ligands

NAG ×2

Metals

_CA ×4

Waters ×436

* Residue conservation analysis

PDB id:

4cel

Name:

Hydrolase

Title:

Active-site mutant d214n determined at ph 6.0 with no ligand bound in the active site

Structure:

1,4-beta-d-glucan cellobiohydrolase i. Chain: a, b. Fragment: catalytic domain, residues 1 - 434. Synonym: exoglucanase. Engineered: yes. Mutation: yes

Source:

Hypocrea jecorina. Organism_taxid: 51453. Strain: qm 9414. Variant: vtt-d-93201. Gene: cbh1. Expressed in: hypocrea jecorina. Expression_system_taxid: 51453.

Resolution:

2.20Å R-factor: 0.206 R-free: 0.239

Authors:

C.Divne,J.Stahlberg,T.A.Jones

Key ref:

J.Ståhlberg et al. (1996). Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei. J Mol Biol, 264, 337-349. PubMed id: 8951380 DOI: 10.1006/jmbi.1996.0644

Date:

24-Aug-96 Release date: 12-Mar-97

Protein chains

P62694  (GUX1_HYPJE) -  Exoglucanase 1 from Hypocrea jecorina

Seq: 513 a.a.

Struc: 434 a.a.*

* PDB and UniProt seqs differ at 3 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: 10.1006/jmbi.1996.0644

J Mol Biol 264:337-349 (1996)

PubMed id: 8951380

Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei.

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

ABSTRACT

The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated by changing these residues to their isosteric amide counterparts. Three mutants, E212Q, D214N and E217Q, were constructed and expressed in T. reesei. All three point mutations significantly impair the catalytic activity of the enzyme, although all retain some residual activity. On the small chromophoric substrate CNP-Lac, the kcat values were reduced to 1/2000, 1/85 and 1/370 of the wild-type activity, respectively, whereas the KM values remained essentially unchanged. On insoluble crystalline cellulose, BMCC, no significant activity was detected for the E212Q and E217Q mutants, whereas the D214N mutant retained residual activity. The consequences of the individual mutations on the active-site structure were assessed for two of the mutants, E212Q and D214N, by X-ray crystallography at 2.0 A and 2.2 A resolution, respectively. In addition, the structure of E212Q CBHI in complex with the natural product, cellobiose, was determined at 2.0 A resolution. The active-site structure of each mutant is very similar to that of the wild-type enzyme. In the absence of ligand, the active site of the D214N mutant contains a calcium ion firmly bound to Glu212, whereas that of E212Q does not. This supports our hypothesis that Glu212 is the charged species during catalysis. As in the complex of wild-type CBHI with bound o-iodobenzyl-1-thio-beta-D-glucoside, cellobiose is bound to the two product sites in the complex with E212Q. However, the binding of cellobiose differs from that of the glucoside in that the cellobiose is shifted away from the trio of catalytic residues to interact more intimately with a loop that is part of the outer wall of the active site.

Selected figure(s)

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 reprinted by permission from Elsevier: J Mol Biol (1996, 264, 337-349) copyright 1996.

Figures were selected by an automated process.