PMID:14767072

Citation	Rothman, SC, Voorhies, M and Kirsch, JF (2004) Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase. Protein Sci. 13:763-72
Abstract	The Escherichia coli aspartate (AATase) and tyrosine (TATase) aminotransferases share 43% sequence identity and 72% similarity, but AATase has only 0.08% and 0.01% of the TATase activities (k(cat)/K(m)) for tyrosine and phenylalanine, respectively. Approximately 5% of TATase activity was introduced into the AATase framework earlier both by rational design (six mutations, termed HEX) and by directed evolution (9-17 mutations). The enzymes realized from the latter procedure complement tyrosine auxotrophy in TATase deficient E. coli. HEX complements even more poorly than does wild-type AATase, even though the (k(cat)/K(m)) value for tyrosine exhibited by HEX is similar to those of the enzymes found from directed evolution. HEX, however, is characterized by very low values of K(m) and K(D) for dicarboxylic ligands, and by a particularly slow release for oxaloacetate, the product of the reaction with aspartate and a TCA cycle intermediate. These observations suggest that HEX exists largely as an enzyme-product complex in vivo. HEX was therefore subjected to a single round of directed evolution with selection for complementation of tyrosine auxotrophy. A variant with a single amino acid substitution, A293D, exhibited substantially improved TATase function in vivo. The A293D mutation alleviates the tight binding to dicarboxylic ligands as K(m)s for aspartate and alpha-ketoglutarate are >20-fold higher in the HEX + A293D construct compared to HEX. This mutation also increased k(cat)/K(m)(Tyr) threefold. A second mutation, I73V, elicited smaller but similar effects. Both residues are in close proximity to Arg292 and the mutations may function to modulate the arginine switch mechanism responsible for dual substrate recognition in TATases and HEX.
Links	PubMed PMC2286728 Online version:10.1110/ps.03117204
Keywords	Amino Acids/genetics; Amino Acids/metabolism; Aspartate Aminotransferases/genetics; Aspartate Aminotransferases/metabolism; Aspartic Acid/genetics; Aspartic Acid/metabolism; Cell Division/genetics; Cloning, Molecular; DNA Shuffling; Directed Molecular Evolution; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Deletion; Kinetics; Models, Chemical; Molecular Structure; Mutagenesis, Site-Directed/genetics; Phenylalanine/genetics; Phenylalanine/metabolism; Point Mutation/genetics; Protein Engineering; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Substrate Specificity; Sucrose/chemistry; Transformation, Bacterial; Tyrosine Transaminase/genetics; Tyrosine Transaminase/metabolism; Viscosity
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