Cystathionine beta-lyase

Cystathionine beta-lyase (EC 4.4.1.8), also commonly referred to as CBL or β-cystathionase, is an enzyme that primarily catalyzes the following α,β-elimination reaction^[1]

Thus, the substrate of this enzyme is L-cystathionine, whereas its 3 products are homocysteine, pyruvate, and ammonia.^[2][3][4]

Found in

Most of the enzyme's catalytic site residues are conserved amongst the enzymes involved in the transsulfuration pathway.^[6] Other members include cystathionine gamma-synthase, cystathionine gamma-lyase, and methionine gamma lyase.^[9][10] 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.^[11][12]

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.^[6][13] This domain contains residues that interact with the active site of the neighboring subunit to facilitate substrate and cofactor binding.^[4]

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.^[3][4]

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.^[4]

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.^[3] 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.^[14]

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.^[15]

Mechanism

As shown in the

The released lysine can now abstract the proton from the C^α and form a quinoid intermediate, which is facilitated by the delocalization of the negative charge over PLP's conjugated p-system.^[14] Subsequently, the protonation of S^γ induces C^β-S^γ bond cleavage, thereby releasing homocysteine^[3][13]

The external aldimine is displaced by the nucleophilic attack of the lysine, regenerating the catalytically active internal aldimine and releasing dehydroalanine.^[4] Lastly, the enamine tautomerizes into an imine that undergoes hydrolytic deamination to form pyruvate and ammonia.^[16]

Inhibition

Plant and bacterial cystathionine beta-lyases are inhibited by the

Excess addition of cystathionine prevented the inactivation of the enzyme, suggesting that AVG acts as a

Through kinetic methods and X-ray crystallography, a time-dependent, slow-binding inhibition was observed. It is believed that the inhibitor binds to the enzyme in a similar way as the substrate; however, after the abstraction of the α-proton, the reaction proceeds to create an inactive ketimine PLP derivative.^[18]

Evolution

Arabidopsis cystathionine beta-lyase possesses 22% homology with its Escherichia coli counterpart and even higher homology (between 28% and 36%) with cystathionine γ-synthase from plant and bacterial sources and cystathionine γ-lyase from Saccharomyces cerevisiae.^[19] All of these enzymes are involved in the Cys/Met biosynthetic pathway and belong to the same class of PLP-dependent enzymes, suggesting that these enzymes were derived from a common ancestor.^[6][20]

Industrial relevance

Cystathionine beta-lyase catalyzes the production of homocysteine, a direct precursor to methionine. Methionine is an essential amino acid for bacteria that is required for protein synthesis and the synthesis of S-adenosylmethionine; thus, the amino acid is directly linked to DNA replication. Because of its necessity in DNA replication, inhibition of cystathionine beta-lyase is an attractive antibiotic target.^[21] Furthermore, the enzyme is absent in humans, decreasing the chance of harmful and unwanted side effects.^[22]

Studies have linked the anti-fungal activity of several anti-fungal agents to the inhibition of cystathionine beta-lyase; however, other studies have not observed enzyme inhibition by these. Further research is needed to characterize the full extent cystathionine beta-lyase inhibition has on microbial and fungal growth.^[21]

References