1ZI9

Crystal Structure Analysis of the dienelactone hydrolase (E36D, C123S) mutant- 1.5 A

1ZI9 の概要

• エントリーDOI: 10.2210/pdb1zi9/pdb
• 関連するPDBエントリー: 1DIN 1GGV 1ZI6 1ZI8 1ZIC 1ZIX 1ZIY 1ZJ4 1ZJ5
• 分子名称: Carboxymethylenebutenolidase, SULFATE ION, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: alpha and beta proteins, 3-d structure, serine esterase, hydrolase, aromatic hydrocarbons, catabolism
• 由来する生物種: Pseudomonas putida
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 25946.16

構造登録者

Kim, H.-K.,Liu, J.-W.,Carr, P.D.,Ollis, D.L. (登録日: 2005-04-27, 公開日: 2005-07-05, 最終更新日: 2023-10-25)

主引用文献

Kim, H.K.,Liu, J.W.,Carr, P.D.,Ollis, D.L.
Following directed evolution with crystallography: structural changes observed in changing the substrate specificity of dienelactone hydrolase.
Acta Crystallogr.,Sect.D, 61:920-931, 2005

PubMed Abstract: The enzyme dienelactone hydrolase (DLH) has undergone directed evolution to produce a series of mutant proteins that have enhanced activity towards the non-physiological substrates alpha-naphthyl acetate and p-nitrophenyl acetate. In terms of steady-state kinetics, the mutations caused a drop in the K(m) for the hydrolysis reaction with these two substrates. For the best mutant, there was a 5.6-fold increase in k(cat)/K(m) for the hydrolysis of alpha-naphthyl acetate and a 3.6-fold increase was observed for p-nitrophenyl acetate. For alpha-naphthyl acetate the pre-steady-state kinetics revealed that the rate constant for the formation of the covalent intermediate had increased. The mutations responsible for the rate enhancements map to the active site. The structures of the starting and mutated proteins revealed small changes in the protein owing to the mutations, while the structures of the same proteins with an inhibitor co-crystallized in the active site indicated that the mutations caused significant changes in the way the mutated proteins recognized the substrates. Within the active site of the mutant proteins, the inhibitor was rotated by about 180 degrees with respect to the orientation found in the starting enzyme. This rotation of the inhibitor caused the displacement of a large section of a loop on one side of the active site. Residues that could stabilize the transition state for the reaction were identified.

PubMed: 15983415
DOI: 10.1107/S0907444905009042

実験手法

X-RAY DIFFRACTION (1.5 Å)