Engineering an F-Amine Dehydrogenase for Efficient and Sustainable Biocatalytic Synthesis of a Chiral PDE10 Inhibitor Precursor

Abstract (R)–4-(1H-indol-3-yl)butan-2-amine ((R)-4IBA), encompassing a nitrogen heterocycle and a chiral primary amino group, is a highly appealing precursor in the pharmaceutical domain. The direct amination of the readily available prochiral ketone 4-(1H-indol-3-yl)butan-2-one (4IBO) catalyzed by amine dehydrogenases (AmDHs) represents a green and sustainable synthetic route for (R)-4IBA, as it consumes only ammonia and releases water as the sole byproduct. However, AmDH-based biocatalytic synthesis of (R)-4IBA remains challenging due to the absence of available AmDH biocatalysts. Here, we engineered the phenylalanine AmDH originating from Bacillus badius (F-BbAmDH) for the biocatalytic synthesis of (R)-4IBA. By combining combinatorial active-site saturation test (CAST) and focused rational iterative site-specific mutagenesis (FRISM) strategies, a quintuple mutant M3-1 (V144G/L306V/V309G/Q308A/T124A) was identified with a 56.6-fold increased kcat/Km value toward substrate 4IBO. The practical application potential of M3-1 was verified in a preparative-scale biosynthesis of (R)-4IBA with a substrate fed-batch strategy. A total of 109.6 mM 4IBO was effectively transformed with 91.7% conversion after four times fed-batch operations, and the product (R)-4IBA was prepared in 82.4% isolated yield and up to 99% ee. Our work presents the first instance of the preparation of (R)-4IBA through enzyme-based sustainable biosynthesis.