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J Biol Chem, Vol. 274, Issue 3, 1320-1325, January 15, 1999

Conversion of Tyrosine Phenol-lyase to Dicarboxylic Amino Acid -Lyase, an Enzyme Not Found in Nature

From the Biochemisches Institut der Universität Zürich, CH-8057 Zürich, Switzerland

Tyrosine phenol-lyase (TPL), which catalyzes the -elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the -family of vitamin B₆-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the -elimination activity toward dicarboxylic amino acids at least 10⁴-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (k_cat = 0.21 s¹) was two times higher than that toward L-tyrosine. -Elimination and transamination as a minor side reaction (k_cat = 0.001 s¹) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid -lyase is an enzyme not found in nature.

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