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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.

Reductive amination of carbonyl compounds constitutes one of the most efficient ways to rapidly construct chiral and achiral amine frameworks. Imine reductase (IRED) biocatalysts represent a versatile family of enzymes for amine synthesis through NADPH‐mediated imine reduction. The reductive aminases (RedAms) are a subfamily of IREDs that were recently shown to catalyze imine formation as well as imine reduction. Herein, a diverse library of novel enzymes were expressed and screened as cell‐free lysates for their ability to facilitate reductive amination to expand the known suite of biocatalysts for this transformation and to identify more enzymes with potential industrial applications. A range of ketones and amines were examined, and enzymes were identified that were capable of accepting benzylamine, pyrrolidine, ammonia, and aniline. Amine equivalents as low as 2.5 were employed to afford up to >99 % conversion, and for chiral products, up to >98 % ee could be achieved. Preparative‐scale reactions were conducted with low amine equivalents (1.5 or 2.0) of methylamine, allylamine, and pyrrolidine, achieving up to >99 % conversion and 76 % yield.

A major challenge in chemical synthesis is to develop catalytic systems that convert simple molecules to complex high-value products. Often these valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Of great interest are enantioenriched amine diastereomers, which are prevalent in pharmaceuticals and agrochemicals, 1 yet their preparation often relies on low-e ciency multi-step synthesis. 2 Herein, we report the discovery and characterisation of a multi-functional biocatalyst, which operates using a previously unreported conjugate reduction-reductive amination mechanism. This enzyme (pIR-120), identi ed within a metagenomic imine reductase (IRED) collection 3 and originating from an unclassi ed Pseudomonas species, possesses an unusual active site architecture that facilitates an amine-activated conjugate alkene reduction followed by reductive amination. This enzyme enables the coupling of a broad selection of α,β-unsaturated carbonyls with amines for the e cient preparation of enantioenriched amine diastereomers. Moreover, employing a racemic substrate partner or conjugated dienyl-ketone provides a means of controlling additional stereocentres using the single catalyst. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by pIR-120 which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.

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