Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C-C bond cleavage

Abstract

The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cβ-Cγ bond of L-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon-carbon bond. As for most other pyridoxal 5'-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-L-tyrosine followed by flash-cooling. The X-ray structures, determined at ~2.0 Å resolution, reveal two quinonoid geometries: "relaxed" in the open and "tense" in the closed state of the active site. The "tense" state is characterized by changes in enzyme contacts made with the substrate's phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate's Cβ-Cγ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the α-aminoacrylate intermediate with bound phenol. In this state, the enzyme-ligand contacts remain almost exactly the same as in the "tense" quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.

Scheme 1. β-Elimination Reaction of l-Tyr Catalyzed by TPL

Chart 1. 3-Fluoro-l-tyrosine

Chart 2. Quinonoid Intermediate with 3-Fluoro-l-tyrosine with the Corresponding Atom-Numbering Scheme

Figure 1

Quinonoid intermediates of the Cβ–Cγ bond cleavage trapped in crystal structures (stereo views). The σ_A-weighted |F_o| – |F_c| omit electron density maps contoured at 3.0σ are in blue. Residues from the large domain are colored in orange, those from the small domain are in pink, and residues from the adjacent subunit are depicted in blue and labeled with an asterisk. Hydrogen bonds are denoted by dashed lines. (a) Open active site (B) of Y71F TPL with the “relaxed” 3-F-Tyr quinonoid. (b) One of the four closed active sites of F448H TPL with the “tense” 3-F-Tyr quinonoid. (c) The disordered active site (A) of Y71F TPL occupied mostly by the “tense” (gray; 0.67 occupancy) and partially by the “relaxed” 3-F-Tyr quinonoid (yellow; 0.33 occupancy). Only the closed active-site conformation could be modeled; a green hashed cylinder indicates a close contact between Phe448 and the “tense” quinonoid.

Figure 2

(a) Geometry of a 3-F-Tyr quinonoid molecule in the “tense” conformation observed in the closed active sites of F448H and Y71F TPL. (b) A stereo view of a 3-F-l-Tyr quinonoid in a closed active site of F448H refined using the standard (yellow) and relaxed (gray) geometrical restraints. The undistorted quinonoid geometry (thin black lines) is shown for comparison. The σ_A-weighted |F_o| – |F_c| omit electron density map is shown at 3.0σ.

Figure 3

Ligands modeled in the disordered active site of Y71F TPL. A stereo view with the “relaxed” (yellow) and “tense” (gray) quinonoid molecules superimposed with the corresponding σ_A-weighted |F_o| – |F_c| omit electron density maps contoured at 3.0σ.

Figure 4

Stereo view of the closed active site of the wild-type TPL with bound PNO. The σ_A-weighted |F_o| – |F_c| omit electron density maps are contoured at 3.0σ. Residues are colored as in Figure 1. Hydrogen bonds are denoted by dashed lines. The alanine quinonoid, the phosphate anion, and the Wat2 solvent molecule were modeled with 0.5 occupancy.

Figure 5

Protein–substrate interactions during the three intermediate steps of the Cβ–Cγ bond cleavage observed in crystal structures. Hydrogen bonds are shown as dashed lines. A short van der Waals contact between the side chain of Phe448 and the substrate phenolic group is denoted by a hashed line. Label colors correspond to residue colors in Figure 1.