Download MendeleyRedesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin.
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.(2013) Protein Eng Des Sel 26: 25-33
- PubMed: 23012440
- DOI: https://doi.org/10.1093/protein/gzs065
- Primary Citation of Related Structures:
4E3Q, 4E3R - 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.
Organizational Affiliation:
Pfizer Worldwide Research and Development, Eastern Point Road, Groton, CT 06340, USA. [email protected]
