Cystathionine beta-lyase (), also commonly referred to as CBL or β-cystathionase, is an enzyme that primarily catalyzes the following α,β-elimination reaction
Thus, the substrate of this enzyme is L-cystathionine, whereas its 3 products are homocysteine, pyruvate, and ammonia.
Found in plants, bacteria, and yeast, cystathionine beta-lyase is an essential part of the methionine biosynthesis pathway as homocysteine can be directly converted into methionine by methionine synthase. The enzyme belongs to the γ-family of PLP-dependent enzymes due to its use of a pyridoxal-5'-phosphate (PLP) cofactor to cleave cystathionine. The enzyme also belongs to the family of lyases, specifically the class of carbon-sulfur lyases. The systematic name of this enzyme class is L-cystathionine L-homocysteine-lyase (deaminating; pyruvate-forming). This enzyme participates in 5 metabolic pathways: methionine metabolism, cysteine metabolism, selenoamino acid metabolism, nitrogen metabolism, and sulfur metabolism.
Structure
Cystathionine beta-lyase is a tetramer composed of identical subunits, and is constructed as a dimer of dimers, each associated with one molecule of PLP bound to the catalytic site by a lysine residue. The dimer is formed by two monomers associated through several electrostatic, hydrogen bonding, and hydrophobic interactions, whereas the tetramer is stabilized through interactions between the N-terminal domains and key α-helices.
Most of the enzyme's catalytic site residues are conserved amongst the enzymes involved in the transsulfuration pathway. Other members include cystathionine gamma-synthase, cystathionine gamma-lyase, and methionine gamma lyase. Additionally, these structures exhibit a type I fold and belong to the aspartate aminotransferase (AAT) family, characterized by homodimers with dihedral symmetry and active sites composed of residues belonging to adjacent subunits.
Monomer
The cystathionine beta-lyase monomer consists of three functionally and structurally distinct domains:
N-terminal domain
Composed of three α-helices and one beta-strand that contribute to the formation of the quaternary structure. This domain contains residues that interact with the active site of the neighboring subunit to facilitate substrate and cofactor binding.
PLP-binding domain
Contains most of the catalytically relevant residues on the enzyme. It is composed of α-helices and β-sheets with a distinct parallel seven-stranded β-sheet. These sheets form a curved structure around the PLP-binding helix. PLP is covalently attached to a lysine residue at the C-terminus of the sheet.
C-terminal domain
Smallest domain on the enzyme, which is attached to the PLP-binding domain by a long, kinked α-helix. The domain is structured into four-stranded antiparallel β-sheet with neighboring helices.
Catalytic site
Aside from being bound to a lysine residue, PLP is fixed within the substrate binding site of the enzyme through various interactions with catalytic residues. Amine- and hydroxyl-containing residues are located in hydrogen bonding distance to the four phosphate oxygens. This phosphate group is considered to be the main contributor to securing PLP in the active site. Additionally, residues neighboring the pyridine nitrogen in PLP help stabilize its positive charge, thereby increasing its electrophilic character.
The aromatic ring in PLP is fixed in place by an almost coplanar tyrosine residue. It is believed that this configuration increases the electron sink character of the cofactor. These stacking interactions between PLP and aromatic side chains can be found in most PLP-dependent enzymes as it plays an important role in catalyzing the reaction by facilitating transaldimination.
Mechanism
As shown in the mechanism below, cystathionine beta-lyase facilitates the S-C bond cleavage in cystathionine with the use of a PLP cofactor bounded to a catalytic lysine residue. Initially, a deprotonated amino group is needed to perform the transaldimination reaction. Given that the pH optimum for the enzyme is between 8.0 and 9.0, a tyrosine residue in the catalytic pocket exists as a phenolate, which abstracts a proton from the α-amino group of the substrate. In the next step, the deprotonated amine undergoes a nucleophilic attack and displaces the lysine to form a Schiff base, forming an internal aldimine.
The released lysine can now abstract the proton from the Cα and form a quinoid intermediate, which is facilitated by the delocalization of the negative charge over PLP's conjugated p-system. Subsequently, the protonation of Sγ induces Cβ-Sγ bond cleavage, thereby releasing homocysteine