title: A high-throughput assay for screening L- or D-amino acid specific aminotransferase mutant libraries.
abstract: Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the L-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either L- or D-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 D-amino acid aminotransferase displaying a decrease in k(cat)/K(M) of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate L-leucine than L-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.
authors.affiliation: Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Curtis J W
authors.initials: CJ
authors.lastname: Walton
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Chica
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation; D015202:Protein Engineering; D013379:Substrate Specificity; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D007656:Ketoglutaric Acids; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase
keywords: Coupled enzyme assay; Enzyme kinetics; Mutant library screening; Protein engineering; Saturation mutagenesis