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Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase
Author(s) -
Rothman Steven C.,
Voorhies Mark,
Kirsch Jack F.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03117204
Subject(s) - tyrosine , complementation , biochemistry , auxotrophy , directed evolution , enzyme , stereochemistry , tyrosine aminotransferase , biology , amino acid , chemistry , escherichia coli , mutant , gene , enzyme inducer
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 α‐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.