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Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin.
Midelfort, Kumar, Han, Karmilowicz, McConnell, Gehlhaar, Mistry, Chang, Anderson, Villalobos, Minshull, Govindarajan, Wong.
ATUM (DNA2.0), Pfizer
Protein Engineering, Design & Selection 2013, 26(1):25-33
Gene names: Vfat. Host systems: Escherichia coli[Bacterial]. Gene species: Paracoccus denitrificans[Bacterial], Rhodobacter sphaeroides[Bacterial], Vibrio fluvialis[Bacterial]. Optimized: Yes. Protein activity: Yes. Vectors: pET28b. Protein engineering / Variant library: Yes.
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,
Comments: Researchers from Pfizer and DNA2.0 engineered an optimized Vfat enzyme with a 60-fold increase in initial reaction velocity. Using ProteinGPS technology and gene synthesis from DNA2.0, less than 450 variants were needed for screening and successful protein engineering.