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 PLP-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. In spite of 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 Å and 1.95 Å resolution, respectively, by cryocrystallography. 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 β-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton.

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

“…A mixture of a quinonoid species and other intermediates has been reported in the 2.9-Å crystal structure of PLPdependent serine hydroxymethyltransferase, in which retro-aldol cleavage of the Cα-Cβ bond rather than proton abstraction from Cα generates a quinonoid intermediate (32). Quinonoid intermediates have also been reported in the structures of tyrosine phenol lyase in the presence of substrate analogs (33) and in tryptophan synthase in the presence of a substrate mimic (34). Although the quinonoid species obtained with a substrate mimic in the tryptophan synthase structure is not directly comparable to the serine-derived intermediate in dCBS, it exhibits a similar Cα geometry.…”

Crystallographic Snapshots of Tyrosine Phenol-lyase Show That Substrate Strain Plays a Role in C–C Bond Cleavage

“…The corresponding quinonoid geometry is characterized by the sp 2 -hybridized Cα and Cγ atoms, each in a trigonal planar environment, and the phenolic moiety rotated with respect to the Cα–Cβ bond (torsion angle Cα–Cβ–Cγ–Cδ1) by 73° (Figure S1 in the Supporting Information). All H-bonds and salt bridges with the enzyme observed for the quinonoid intermediates formed with l -Ala or l -Met(11) are also found in the open active site of the 3-F-Tyr quinonoid (Figure 1a). Additionally, the hydroxyl group of the 3-F-Tyr quinonoid complex forms H-bonds with two water molecules, while the fluorine atom is H-bonded to only one of them.…”

The role of substrate strain in the mechanism of the carbon–carbon lyases

“…However, no structures of substrate or inhibitor complexes of TIL have been obtained to date. C. freundii TPL crystallizes readily under a range of conditions, and a number of complexes of wild type and mutant TPLs with substrates and inhibitors have been obtained [17,21,45,46]. Thus, the TPL complexes provide the majority of the structural information on the mechanisms of the carbon-carbon lyases.…”

“…Although no structures have been obtained of an external aldimine of TPL, there are a number of structures of quinonoid intermediates of wild-type TPL with L-alanine and L-methionine [45], and Y71F and F448H mutant TPL with 3-F-L-tyrosine [46]. The quinonoid intermediate structures with 3-F-L-tyrosine form either open (relaxed) or closed (tense) conformations.…”