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 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 beta-elimination activity toward dicarboxylic amino acids at least 10(4)-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 (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine. beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) 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 beta-lyase is an enzyme not found in nature.
Mouratou
B
B
Biochemisches Institut der Universität Zürich, CH-8057 Zürich, Switzerland.
Kasper
P
P
Gehring
H
H
Christen
P
P
D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D016971:Citrobacter freundii; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity; D014447:Tyrosine Phenol-Lyase / Q000378:metabolism*
D000600:Amino Acids, Dicarboxylic; D001219:Aspartate Aminotransferases; D014447:Tyrosine Phenol-Lyase