Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase,an enzyme not found in nature

Tyrosine phenol-lyase (TPL), which catalyzes the beta-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 alpha-family of vitamin B-6-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic am ino acids, two amino acid residues of AspAT, thought to be important for th e 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 sub strates and is conserved among all known AspATs. Arg-100 in TPL was found t o correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the beta- elimination activity to ward dicarboxylic amino acids at least 10(4)-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) w ere 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 s(-1)) was two times higher than that toward L-tyrosine. beta -Elimination and transamination as a minor side reaction (k(cat) = 0.001 s( -1)) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarb oxylic amino acids as substrates without significant change in its reaction specificity dicarboxylic amino acid beta-lyase is an enzyme not found in n ature.