Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase

doi:10.1110/ps.03117204

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Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase

Steven C. Rothman, Mark Voorhies and Jack F. Kirsch

University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley, California 94720-3206, USA

(RECEIVED April 3, 2003; FINAL REVISION October 30, 2003; ACCEPTED October 31, 2003)

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. Approximately5% of TATase activity was introduced into the AATase frameworkearlier both by rational design (six mutations, termed HEX)and by directed evolution (9–17 mutations). The enzymesrealized from the latter procedure complement tyrosine auxotrophyin TATase deficient E. coli. HEX complements even more poorlythen does wild-type AATase, even though the (k_cat/K_m) valuefor tyrosine exhibited by HEX is similar to those of the enzymesfound from directed evolution. HEX, however, is characterizedby very low values of K_m and K_D for dicarboxylic ligands, andby a particularly slow release for oxaloacetate, the productof the reaction with aspartate and a TCA cycle intermediate.These observations suggest that HEX exists largely as an enzyme–productcomplex in vivo. HEX was therefore subjected to a single roundof directed evolution with selection for complementation oftyrosine 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 dicarboxylicligands as K_ms for aspartate and ${alpha}$ -ketoglutarate are >20-foldhigher in the HEX + A293D construct compared to HEX. This mutationalso increased k_cat/K_m^Tyr threefold. A second mutation, I73V,elicited smaller but similar effects. Both residues are in closeproximity to Arg292 and the mutations may function to modulatethe arginine switch mechanism responsible for dual substraterecognition in TATases and HEX.

Keywords: aminotransferase; protein engineering; directed evolution; pyridoxal phosphate; substrate specificity

Abbreviations: AATase, aspartate aminotransferase (EC 2.6.1.1) • TATase, tyrosine aminotransferase (EC 2.6.1.5) • HEX, a mutant of AATase with the substitutions V39L/K41Y/T47I/N69L/T109S/N297S • 8-2, a directly evolved mutant of AATase with the substitutions A13T/A26V/N69S/G72D/S139G/T167A/R282C/A293V/N297S/N339S/A381V/N396D/A398V • HO-HxoDH, 2-hydroxyisocaproate (2-hydroxy-4-methyl-pentanoate) dehydrogenase (EC 1.1.1.-) • MDH, malate dehydrogenase (EC 1.1.1.37) • PLP, pyridoxal 5'-phosphate • PMP, pyridoxamine 5'-phosphate • ${alpha}$ KG, ${alpha}$ -ketoglutarate • HPP, hydroxyphenylpyruvate • PP, phenylpyruvate • OAA, oxaloacetate

Reprint requests to: Jack F. Kirsch, University of California, Berkeley, Department of Molecular and Cell Biology, 229 Stanley Hall #3206, Berkeley, CA 94720-3206, USA; e-mail: jfkirsch{at}uclink.berkeley.edu; fax: (510) 642-6368.

Article published online ahead of print. Article and publicationdate are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03117204.

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