Asymmetrically simultaneous synthesis of L-homophenylalanine and N6-protected-2-oxo-6-amino-hexanoic acid by engineered Escherichia coli aspartate aminotransferase.
L-Homophenylalanine (L-HPA) and N(6)-protected-2-oxo-6-amino-hexanoic acid (N(6)-protected-OAHA) can be used as building blocks for the manufacture of angiotensin-converting enzyme inhibitors. To synthesize L-HPA and N(6)-protected-OAHA simultaneously from 2-oxo-4-phenylbutanoic acid (OPBA) and N(6)-protected-L-lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site-directed mutagenesis and their catalytic activities were investigated. Three kinds of N(6)-protected-L-lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70+/-0.01 U/mg protein and 0.67+/-0.02 U/mg protein to 2-amino-6-tert-butoxycarbonylamino-hexanoic acid (BOC-lysine) and 2-amino-6-(2,2,2-trifluoro-acetylamino)-hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC-lysine to L-HPA and 2-oxo-6-tert-butoxycarbonylamino-hexanoic acid (BOC-OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L-HPA and BOC-OAHA.
Lo
Hsueh-Hsia
HH
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan.
Lin
Wei-De
WD
Hsu
Shih-Kuang
SK
Hsu
Wen-Hwei
WH
D000613:Aminobutyrates / Q000737:chemistry / Q000378:metabolism*; D000614:Aminocaproates; D015119:Aminocaproic Acid / Q000737:chemistry; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002208:Caproates / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D009154:Mutation; D053719:Tandem Mass Spectrometry
C548303:2-oxo-6-tert-butoxycarbonylaminohexanoic acid; D000613:Aminobutyrates; D000614:Aminocaproates; D001426:Bacterial Proteins; D002208:Caproates; C014328:2-amino-4-phenylbutyric acid; D001219:Aspartate Aminotransferases; D015119:Aminocaproic Acid