Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site Saturation Mutagenesis (GSSM)

• Published in: Journal of the American Chemical Society 2003-09, Vol.125 (38), p.11476-11477
• Main Authors: DeSantis, Grace, Wong, Kelvin, Farwell, Bob, Chatman, Kelly, Zhu, Zoulin, Tomlinson, Geoff, Huang, Hongjun, Tan, Xuqiu, Bibbs, Lisa, Chen, Pei, Kretz, Keith, Burk, Mark J
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Aminohydrolases - chemistry; Aminohydrolases - genetics; Aminohydrolases - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Hydroxybutyrates - chemical synthesis; Mutagenesis, Site-Directed; Stereoisomerism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja035742h

Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.