Three-Dimensional Structure of Tyrosine Phenol-lyase

Tyrosine phenol-lyase (EC 4.1.99.2) from Citrobacterfreundii has been cloned and the primary sequence deduced from the DNA sequence. From the BrCN digest of the NaBH₄-reduced holoenzyme, five peptides were purified and sequenced. The amino acid sequences of the peptides agreed with the corresponding parts of the tyrosine phenol-lyase sequence obtained from the gene structure. K257 is the pyridoxal 5′-phosphate binding residue. Assisted by the sequence data, the crystal structure of apotyrosine phenol-lyase, a pyridoxal 5–phosphate-dependent enzyme, has been refined to an R-factor of 16.2% at 2.3-Å resolution using synchrotron radiation diffraction data. The tetrameric molecule has 222 symmetry, with one of the axes coincident with the crystallographic 2-fold symmetry axis of the crystal which belongs to the space group P2₁2₁2 with a = 76.0 Å, b = 138.3 Å, and c = 93.5 Å. Each subunit comprises 14 α-helices and 16 β-strands, which fold into a small and a large domain. The coenzyme-binding lysine residue is located at the interface between the large and small domains of one subunit and the large domain of a crystallographically related subunit. The fold of the large, pyridoxal 5′-phosphate binding domain and the location of the active site are similar to that found in aminotransferases. Most of the residues which participate in binding of pyridoxal 5′-phosphate in aminotransferases are conserved in the structure of tyrosine phenol-lyase. Two dimers of tyrosine phenol-lyase, each of which has a domain architecture similar to that found in aspartate aminotransferases, are bound together through a hydrophobic cluster in the center of the molecule and intertwined N-terminal arms.