4E3Q

PMP-bound form of Aminotransferase crystal structure from Vibrio fluvialis

Summary for 4E3Q

• Entry DOI: 10.2210/pdb4e3q/pdb
• Related: 4E3R
• Descriptor: Pyruvate transaminase, SODIUM ION, SULFATE ION, ... (6 entities in total)
• Functional Keywords: aminotransferase, transferase
• Biological source: Vibrio fluvialis
• Total number of polymer chains: 4
• Total formula weight: 213884.61

Authors

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. (deposition date: 2012-03-10, release date: 2012-10-10, Last modification date: 2024-11-27)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.9 Å)