1TOJ

Hydrocinnamic acid-bound structure of SRHEPT mutant of E. coli aspartate aminotransferase

1TOJ の概要

• エントリーDOI: 10.2210/pdb1toj/pdb
• 関連するPDBエントリー: 1TOE 1TOG 1TOI
• 分子名称: Aspartate aminotransferase, HYDROCINNAMIC ACID (3 entities in total)
• 機能のキーワード: aspartate aminotransferase hexamutant, srhept, transferase
• 由来する生物種: Escherichia coli
• 細胞内の位置: Cytoplasm: P00509
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 43975.46

構造登録者

Chow, M.A.,McElroy, K.E.,Corbett, K.D.,Berger, J.M.,Kirsch, J.F. (登録日: 2004-06-14, 公開日: 2004-10-05, 最終更新日: 2023-11-15)

主引用文献

Chow, M.A.,McElroy, K.E.,Corbett, K.D.,Berger, J.M.,Kirsch, J.F.
Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase
Biochemistry, 43:12780-12787, 2004

PubMed Abstract: Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release.

PubMed: 15461450
DOI: 10.1021/bi0487544

実験手法

X-RAY DIFFRACTION (1.9 Å)