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

Abstract

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 B 6 -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 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 (k cat ‫؍‬ 0.21 s ؊1 ) was two times higher than that toward L-tyrosine. ␤-Elimination and transamination as a minor side reaction (k cat ‫؍‬ 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 ␤-lyase is an enzyme not found in nature.The pyridoxal 5Ј-phosphate-dependent enzymes (B 6 enzymes) catalyze a wide variety of reactions in the metabolism of amino acids (1). A comparison of amino acid sequences has shown that the majority of B 6 enzymes belong to the large ␣/␥-superfamily of homologous B 6 enzymes (2, 3). Tyrosine phenol-lyase (TPL) 1 of Citrobacter freundii is a member of the ␣-family. It catalyzes the ␤-elimination of L-tyrosine to produce phenol, pyruvate, and ammonium (Equation 1).A number of amino acids with suitable leaving groups on C␤, such as L-serine and O-acyl-L-serines (4), L-cysteine, S-alkyl-Lcysteines (4, 5), and 3-chloro-L-alanine, are also substrates for ␤-elimination. Moreover, TPL has been found to catalyze markedly slower side reactions, i.e. ␤-replacement reactions (6, 7), racemization of alanine (8, 9), as well as transamination reactions of its substrates L-tyrosine, L-serine, and of the competitive inhibitors L-alanine, L-phenylalanine, and L-m-tyrosine (10).X-ray crystallographic structure analysis has shown the folding pattern of the polypeptide chain of tetrameric TPL from C. freundii to be similar to that of dimeric aspartate aminotransferase (AspAT) (11), which, like TPL, is a member of the ␣-family of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes. Despite their similarity in secondary and tertiary structure, the two enzymes show only low sequence identity, e.g. 23% between TPL of C. freundii and AspAT of Escherichia coli. AspAT catalyzes the reversible transaminatio...

The mechanism of α‐proton isotope exchange in amino acids catalysed by tyrosine phenol‐lyase

“…It was supposed that these nucleophilic substituents interact with an electrophilic group in the active site [5]. Evidence was presented by Mouratou et al [18] that Arg100 occupies a suitable position to perform such an interaction. In the present work we examined the mechanisms of isotopic exchange of a-proton catalyzed by TPL in reactions with L-phenylalanine and L-methionine which may be considered as typical representatives of the two groups of amino acid inhibitors mentioned above.…”

“…Some comments are necessary as to the role played by the interaction of the side group of L-methionine with Arg100 in the considered reactions. For L-aspartic acid the interaction of the distal carboxylic group with Arg100 takes place in the quinonoid intermediate structure [18], but not in the external aldimine. The observed predominant formation of the quinonoid intermediate in the reaction of TPL with L-methionine gives evidence for the presence of a similar interaction of sulfur atom with Arg100, and the observed very low rate of reprotonation evidently reflects the stabilization of the quinonoid intermediate by this interaction.…”

The Molecular Evolution of Pyridoxal‐5′‐Phosphate‐Dependent Enzymes

“…An example for changing the substrate specificity is the conversion of tyrosine phenol-lyase to dicarboxylic amino acid P-lyase (Mouratou et al, 1999). Tyrosine P-lyase is a member of the a family.…”

Conversion of Aspartate Aminotransferase into anl-Aspartate β-Decarboxylase by a Triple Active-site Mutation

“…Among the pyridoxal 5Ј-phosphate-dependent enzymes, aspartate aminotransferase (AspAT) 1 has been converted by multiple active-site mutations into an L-tyrosine aminotransferase (5) and by directed molecular evolution into an L-branched-chain amino acid aminotransferase (7,8). Tyrosine phenol-lyase has been engineered by a double mutation to act as a dicarboxylic-acid ␤-lyase (an enzyme not found in nature) that degrades aspartate to pyruvate, ammonia, and formate (9). However, as yet, no change in the reaction specificity of an enzyme has been reported, with the exception of the conversion of papain into a peptide-nitrile hydratase (10).…”