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Keywords:

  • Aminotransferase;
  • chimera;
  • context dependence;
  • protein/genetic engineering;
  • pyridoxal phosphate;
  • substrate specificity
  • αKG, α-ketoglutarate;
  • AATase, aspartate aminotransferase (EC 2.6.1.1);
  • Grease AATase, a mutant of AATase with the substitutions V39L/K41Y/T47I/N69L;
  • Hca, hydrocinnamate;
  • Hex AATase, a mutant of AATase with the substitutions V39L/K41Y/T47I/N69L/T109S/N297S;
  • 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;
  • retroGrease TATase, a mutant of TATase with the substitutions L39V/Y41K/I47T/L69N;
  • retroHex TATase, a mutant of TATase with the substitutions L39V/Y41K/I47T/L69N/S109T/S297N;
  • Trip AATase, a mutant of AATase with the substitutions V39L/T47I/N69L

Abstract

The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E. coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L). The letters on the left and right of the numbers designate AATase and TATase residues, respectively. The T109S/N297S pair has been investigated previously. The latter group, the “Grease” set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase. The Grease mutations in the AATase framework were found primarily to lower KMs for both aromatic and dicarboxylic substrates. In contrast, retroGrease TATase exhibits lowered kcats for both substrates. The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (kcat/KM) for aspartate over phenylalanine. The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pKas, 6.1 and 9.1. RetroHex was also found to have impaired dimer stability, with a KD for dimer dissociation of 350 nM compared with the wild type KD of 4 nM. Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular. Context dependence and cooperativity are particularly evident in the effects of mutations on kcat/KM(Asp). Effects on kcat/KM(Phe) are more nearly additive and context independent.