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.

A new target region for changing the substrate specificity of amine transaminases

“…ATAs transfer an amino group of a chiral amine compound to a ketone compound using the cofactor pyridoxal 5’-phosphate (PLP) (Supplementary Fig. 1a) with a high turnover rate, stable catalytic activity, broad substrate specificity and excellent stereoselectivity7, which is achieved by a proposed large-binding pocket (L pocket) and small-binding pocket (S pocket) in the substrate-binding site8910111213141516 (Supplementary Fig. 1b).…”

“…Up to now, several structures of S -ATAs9101112 and R -ATAs13141516 have been determined, providing some information about the dual substrate recognition30 of ATAs, which is a unique ability of transaminases to accept both the hydrophilic and hydrophobic substrates in the same active site. In the S -ATAs, a “flipping” arginine residue in a loop near the active site was proposed to play a key role in dual substrate recognition by moving the guanidino group in the active site to interact with the carboxylate of substrates or moving out of the active site to provide a hydrophobic environment9.…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…Furthermore, a residue providing a hydrogen bond to the phenolic oxygen of the cofactor is present in the majority of AT-II enzymes; exceptions include DAPA-AT [8,59] and other enzymes (e.g., [65]), where the hydrogen bond is water-mediated. Hydrogen bonding the phenolic oxygen may be important for modulating the pK a of the internal aldimine (the Schiff base of the PLP with the catalytic lysine) [66] and also the reactivity of PLP [67].…”

Transaminases and their Applications

“…Recently, Midelfort et al evaluated several engineering techniques, like homol ogy modeling, bioinformatics, machine learning, crystal structure, and site-directed mutagenesis of specific sites, to evolve the transaminase from V. fluvialis in selectiv ity and activity for synthesis of (3S,5R)-ethyl-3-amino-5-methyloctanoate, an inter mediate of the imagabalin synthesis [124]. Imagabalin is a candidate for the treatment of anxiety disorder.…”