PDBsum entry: 1oa4

Hydrolase

PDB-id: 1oa4

Contents

Description

Header details

Header records

References

PROCHECK

Protein chain

222 a.a. *

Waters ×117

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

1oa4

Name:

Hydrolase

Title:

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

Structure:

Endo-beta-1,4-glucanase. Chain: a. Fragment: catalytic domain, residues 32-253. Synonym: endoglucanase, cel12a. Engineered: yes

Source:

Streptomyces sp. 11ag8. Organism_taxid: 133452. Expressed in: streptomyces lividans. Expression_system_taxid: 1916

UniProt:

Q9KIH1 (Q9KIH1_9ACTO)

Seq:

Struc:

Seq:

371 a.a.

Struc:

222 a.a.

Key:	PfamA domain
Secondary structure	CATH domain

Resolution:

1.5Å

R-factor:

0.181

R-free:

0.192

Authors:

M.Sandgren,P.J.Gualfetti,A.Shaw,L.S.Gross,M.Saldajeno, A.G.Day,T.A.Jones,C.Mitchinson

Key ref:

M.Sandgren et al. (2003). Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability.. Protein Sci, 12, 848-860. [PubMed id: 12649442] [DOI: 10.1110/ps.0237703]

Date:

28-Dec-02

Release date:

27-Mar-03

Related entries:

1oa2 comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability
1oa3 comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1110/ps.0237703

Protein Sci 12:848-860 (2003)

PubMed id: 12649442

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability.

M.Sandgren, P.J.Gualfetti, A.Shaw, L.S.Gross, M.Saldajeno, A.G.Day, T.A.Jones, C.Mitchinson.

ABSTRACT

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.

Selected figure(s)

Figure 1.
Figure 1. Schematic ribbon diagram. Top, (A) and side (B) views of the H. schweinitzii Cel12A crystal structure, color ramped according to residue number, starting with red at the amino terminus and ending with blue at the carboxyl terminus of the structure. The two ß-sheets in the structure are labeled A and B, with the individual strands labeled (A1-A6 and B1-B9) according to their positions in the two ß-sheets. The structures have side chains drawn for the 14 residues that differ from the T. reesei Cel12A protein sequence. Figures 1 Go-and 3 Go-were prepared using O (Jones et al. 1991), and rendered with Molray (Harris and Jones 2001).

Figure 4.
Figure 4. (A) Interactions and conformational changes close to residue 35 of the fungal GH 12 enzymes from T. reesei (wild type and A35V have carbon atoms colored yellow and goldenrod), and H. schweinitzii (carbons colored gold). Red bubbles indicate contacts in T. reesei A35V Cel12A, blue bubbles in H. schweinitzii Cel12A. (B) Interactions and conformational changes close to residue 34 of the bacterial GH 12 enzymes from S. sp. 11AG8 (carbons colored gold), and S. lividans (carbons colored yellow). Red bubbles indicate contacts in S. lividans CelB2, blue bubbles in S. sp. 11AG8 Cel12A.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2003, 12, 848-860) copyright 2003.

Figures were selected by the author.