Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates

Abstract

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.

SCHEME 1.

Reversible β-elimination reaction of l-tyrosine catalyzed by TPL.

FIGURE 1.

TPL tetramer. Ribbon diagram with different subunits shown in different colors. One catalytic dimer consists of the subunits shown in cyan and blue, while the other is formed from the subunits shown in orange and yellow. PLP and the side chains of the PLP-binding Lys-257 residues are shown as spheres. The view is along the crystallographic 2-fold axis.

SCHEME 2.

Postulated reaction mechanism of β-elimination of l-tyrosine catalyzed by TPL.

FIGURE 2.

Superposition of two TPL subunits that adopt open and closed conformations. The closed conformation is created by the rotation of the small rigid region (residues 19–44, 346–404, 434–456) by about 16° around a hinge connecting it with the large rigid region (residues 1–13, 45–345, 405–422). The stereo view shows TPL-Ala subunits as Cα models superimposed using large rigid regions. The quinonoid molecules are depicted as bold ball-and-stick models. The large and small rigid regions of the closed subunit are shown in orange and pink, respectively, and the flexible parts are shown in yellow. The large and small rigid regions of the open subunit are shown in blue and green, respectively, while the flexible parts are shown in cyan.

FIGURE 3.

Enzyme interactions with the Ala quinonoid intermediate in the closed (A) and open (B) active sites. Stereo views with structures of quinonoid and two water molecules superposed with the corresponding weighted |F_o| - |F_c| electron density omit maps (green) are contoured at the 3.0σ level. Hydrogen bonds are denoted by dashed lines. Carbon atoms of residues belonging to the large rigid region are shown in orange, those from the small rigid region are shown in pink, and the residues from the neighboring subunit are shown in blue and labeled with a star. The alternate conformations of Thr-124, Thr-216, Met-288, and Phe-449 in the open conformation are shown in corresponding pale tones.

FIGURE 4.

Enzyme interactions with the Met quinonoid intermediate in the closed active site. Stereo view with the quinonoid molecule superposed with the corresponding weighted |F_o| - |F_c| electron density omit maps (green) are contoured at the 3.0σ level. Carbon atoms of residues belonging to the large rigid region are shown in orange, those from the small rigid region are shown in pink, and the residues from the neighboring subunit are shown in blue and labeled with a star. Hydrogen bonds are denoted by dashed lines.

SCHEME 3.

Proposed mechanism of alanine racemization catalyzed by TPL.

SCHEME 4.

Concerted mechanism of Cα hydrogen isotope exchange in the external aldimine of methionine.