4E3R

PLP-bound aminotransferase mutant crystal structure from Vibrio fluvialis

4E3R の概要

• エントリーDOI: 10.2210/pdb4e3r/pdb
• 関連するPDBエントリー: 4E3Q
• 分子名称: Pyruvate transaminase, SULFATE ION, SODIUM ION, ... (4 entities in total)
• 機能のキーワード: aminotransferase, transferase
• 由来する生物種: Vibrio fluvialis
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 210173.45

構造登録者

Midelfort, K.S.,Kumar, R.,Han, S.,Karmilowicz, M.J.,McConnell, K.,Gehlhaar, D.K.,Mistry, A.,Chang, J.S.,Anderson, M.,Vilalobos, A.,Minshull, J.,Govindarajan, S.,Wong, J.W. (登録日: 2012-03-10, 公開日: 2012-10-10, 最終更新日: 2024-03-06)

主引用文献

Midelfort, K.S.,Kumar, R.,Han, S.,Karmilowicz, M.J.,McConnell, K.,Gehlhaar, D.K.,Mistry, A.,Chang, J.S.,Anderson, M.,Villalobos, A.,Minshull, J.,Govindarajan, S.,Wong, J.W.
Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin.
Protein Eng.Des.Sel., 26:25-33, 2013

PubMed Abstract: Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related β-ketoesters.

PubMed: 23012440
DOI: 10.1093/protein/gzs065

実験手法

X-RAY DIFFRACTION (1.9 Å)