D-Tryptophan and its derivatives are important precursors of a wide range of indole-containing pharmaceuticals and natural products. Here, we developed a one-pot biocatalytic process enabling the synthesis of D-tryptophans from indoles in good yields and high enantiomeric excess (91% to >99%). Our method couples the synthesis of L-tryptophans catalyzed by Salmonella enterica tryptophan synthase with a stereoinversion cascade mediated by Proteus myxofaciens L-amino acid deaminase and an aminotransferase variant that we engineered to display native-like activity toward D-tryptophan. Our process is applicable to preparative-scale synthesis of a broad range of D-tryptophan derivatives containing electron-donating or -withdrawing substituents at all benzene-ring positions on the indole group.
d‐Phenylalanine derivatives are valuable chiral building blocks for a wide range of pharmaceuticals. Here, we developed stereoinversion and deracemization biocatalytic cascades to synthesize d‐phenylalanine derivatives that contain electron‐donating or ‐withdrawing substituents of various sizes and at different positions on the phenyl ring with a high enantiomeric excess (90 to >99 % ee) from commercially available racemic mixtures or l‐amino acids. These whole‐cell systems couple Proteus mirabilis l‐amino acid deaminase with an engineered aminotransferase that displays native‐like activity towards d‐phenylalanine, which we generated from Bacillus sp. YM‐1 d‐amino acid aminotransferase. Our cascades are applicable to preparative‐scale synthesis and do not require cofactor‐regeneration systems or chemical reducing agents.
Aromatic d-amino acids are key precursors for the production of many small molecule therapeutics. Therefore, the development of biocatalytic methods for their synthesis is of great interest. An enzyme that has great potential as a biocatalyst for the synthesis of d-amino acids is the stereoinverting d-phenylglycine aminotransferase (DPAT) from Pseudomonas stutzeri ST-201. This enzyme catalyzes a unique l to d transamination reaction that produces d-phenylglycine and α-ketoglutarate from benzoylformate and l-glutamate, via a mechanism that is poorly understood. Here, we present the crystal structure of DPAT, which shows that the enzyme folds into a two-domain structure representative of class III aminotransferases. Guided by the crystal structure, we performed saturation mutagenesis to probe the substrate binding pockets of the enzyme. These experiments helped us identify two arginine residues (R34 and R407), one in each binding pocket, that are essential to catalysis. Together with kinetic analyses using a library of amino acid substrates, our mutagenesis and structural studies allow us to propose a binding model that explains the dual l/d specificity of DPAT. Our kinetic analyses also demonstrate that DPAT can catalyze the transamination of β- and γ-amino acids, reclassifying this enzyme as an ω-aminotransferase. Collectively, our studies highlight that the DPAT active site is amenable to protein engineering for expansion of its substrate scope, which offers the opportunity to generate new biocatalysts for the synthesis of a variety of valuable optically pure d-amino acids from inexpensive and abundant l-amino acids.
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