1OA4

Comparison of Family 12 Glycoside Hydrolases and Recruited Substitutions Important for Thermal Stability

1OA4 の概要

• エントリーDOI: 10.2210/pdb1oa4/pdb
• 関連するPDBエントリー: 1OA2 1OA3
• 分子名称: ENDO-BETA-1,4-GLUCANASE (2 entities in total)
• 機能のキーワード: hydrolase, cellulase, cellulose degradation, endoglucanase, glycosyl hydrolase, gh family 12, streptomyces sp 11ag8 cel12a
• 由来する生物種: STREPTOMYCES SP. 11AG8
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 23890.13

構造登録者

Sandgren, M.,Gualfetti, P.J.,Shaw, A.,Gross, L.S.,Saldajeno, M.,Day, A.G.,Jones, T.A.,Mitchinson, C. (登録日: 2002-12-28, 公開日: 2003-03-27, 最終更新日: 2024-11-13)

主引用文献

Sandgren, M.,Gualfetti, P.J.,Shaw, A.,Gross, L.S.,Saldajeno, M.,Day, A.G.,Jones, T.A.,Mitchinson, C.
Comparison of Family 12 Glycoside Hydrolases and Recruited Substitutions Important for Thermal Stability
Protein Sci., 12:848-, 2003

PubMed 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.

PubMed: 12649442
DOI: 10.1110/PS.0237703

実験手法

X-RAY DIFFRACTION (1.5 Å)