Development of an NADPH-dependent homophenylalanine dehydrogenase by protein engineering.
l-Homophenylalanine is a nonproteinogenic amino acid and can be used as a versatile pharmaceutical intermediate. Production of l-homophenylalanine involves amination of the keto acid precursor 2-oxo-4-phenylbutyric acid (2-OPBA), which can be accomplished by bioenzymatic processes. Current biocatalysts for this reaction include transaminases and NADH-dependent phenylalanine dehydrogenases, which are not optimal for metabolic engineering of whole-cell biocatalysis. Here, we report the development of an NADPH-dependent homophenylalanine dehydrogenase by engineering the NADPH-dependent glutamate dehydrogenase (GDH) from Escherichia coli, which provides a new tool for in vitro catalysis and in vivo metabolic engineering. We took a stepwise substrate walking strategy: the first round directed evolution switched GDH's substrate specificity from its natural substrate 2-ketoglutarate to the intermediate target phenylpyruvate, which has similar structure as 2-OPBA; and the second round further improved the enzyme's catalytic efficiency toward the final target 2-OPBA. Compared to wild type GDH, the catalytic efficiency (kcat/Km) of the final mutant was ∼100 fold higher for 2-OPBA and ∼3000 fold lower for the original substrate 2-ketoglutarate. When overexpressed in E. coli, the engineered GDH aminated 2-OPBA to l-homophenylalanine more effectively than the transaminases and NADH-dependent phenylalanine dehydrogenase, possibly because it utilizes the strong anabolic driving force NADPH under aerobic condition.
Department of Chemical and Biomolecular Engineering ‡The Molecular Biology Institute §Department of Chemistry and Biochemistry, ∥Institute of Genomics and Proteomics, University of California, Los Angeles , Los Angeles, California 90095, United States.
Han
H
Li
James C
JC
Liao
D000480:Alkynes / Q000737:chemistry / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000613:Aminobutyrates / Q000378:metabolism; D055162:Biocatalysis; D002087:Butyrates / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D060847:Metabolic Engineering; D009249:NADP / Q000378:metabolism*; D010667:Phenylpyruvic Acids / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering
C513892:2-oxo-4-phenyl-3-butynoate; D000480:Alkynes; D000613:Aminobutyrates; D002087:Butyrates; D007656:Ketoglutaric Acids; D010667:Phenylpyruvic Acids; D009249:NADP; C014328:2-amino-4-phenylbutyric acid; D000594:Amino Acid Oxidoreductases; C073215:phenylalanine dehydrogenase (NAD); D018086:Glutamate Dehydrogenase (NADP+); C031606:phenylpyruvic acid