Creation of a Broad-Range and Highly Stereoselective d-Amino Acid Dehydrogenase for the One-Step Synthesis of d-Amino Acids

• Published in: Journal of the American Chemical Society Vol. 128; no. 33; pp. 10923 - 10929
• Main Authors: Vedha-Peters, Kavitha, Gunawardana, Manjula, Rozzell, J. David, Novick, Scott J
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 23.08.2006; Amer Chemical Soc
• Subjects: Amino Acids - chemical synthesis; Chemistry; Chemistry, Multidisciplinary; Corynebacterium glutamicum - enzymology; D-Amino-Acid Oxidase - chemistry; D-Amino-Acid Oxidase - genetics; D-Amino-Acid Oxidase - metabolism; Exact sciences and technology; General and physical chemistry; Molecular Structure; Mutagenesis, Site-Directed; Niacinamide - chemistry; Physical Sciences; Science & Technology; Substrate Specificity; OXIDATION; ENZYME; PSEUDOMONAS-AERUGINOSA; DIAMINOPIMELATE D-DEHYDROGENASE; ESCHERICHIA-COLI; D-PROLINE; BACILLUS-SPHAERICUS; DIRECTED EVOLUTION; D-ALANINE OXIDASE; CORYNEBACTERIUM-GLUTAMICUM; D-Amino-acid dehydrogenase; Enzyme; Aliphatic compound; Active site; Selectivity; Branched compound; Cofactor; Biocatalyst; Chemical reduction; Stereoselectivity; Ammonia; Screening; Amination; Mutagenesis; Aminoacid; Diaminopimelate dehydrogenase; Enantiomeric excess; Aromatic compound; Nicotinamide; Oxidoreductases; Ketoacid; Mutation; Chemical synthesis; Enantiomer(-)
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0603960

Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective d-amino acid dehydrogenase. This new enzyme is capable of producing d-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate d-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity toward the d-enantiomer (95 to >99% ee) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for d-amino acid synthesis.