title: Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin.
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.
authors.affiliation: Pfizer Worldwide Research and Development, Eastern Point Road, Groton, CT 06340, USA. Midelfort@msoe.edu
authors.forename: Katarina S
authors.initials: KS
authors.lastname: Midelfort
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Seungil
authors.initials: S
authors.lastname: Han
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Karmilowicz
authors.forename: Kevin
authors.initials: K
authors.lastname: McConnell
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Gehlhaar
authors.forename: Anil
authors.initials: A
authors.lastname: Mistry
authors.forename: Jeanne S
authors.initials: JS
authors.lastname: Chang
authors.forename: Marie
authors.initials: M
authors.lastname: Anderson
authors.forename: Alan
authors.initials: A
authors.lastname: Villalobos
authors.forename: Jeremy
authors.initials: J
authors.lastname: Minshull
authors.forename: Sridhar
authors.initials: S
authors.lastname: Govindarajan
authors.forename: John W
authors.initials: JW
authors.lastname: Wong
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D002210:Caprylates / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D002210:Caprylates; D000637:Transaminases; C583768:imagabalin; C031492:octanoic acid