CGL

TgCGL is a PLP-dependent enzyme that catalyzes the terminal step of the reverse transsulfuration pathway for cysteine biosynthesis in Toxoplasma gondii, acting downstream of TgCBS to hydrolyze cystathionine to cysteine (PMID:32887901). Recombinant TgCGL almost exclusively performs α,γ-elimination of l-cystathionine, with only marginal reactivity toward l-cysteine, a near-exclusive substrate specificity dictated by active-site residues Ser77 and Asn360 that diverge from the human enzyme: mutation Asn360Ser broadens activity to include both α,β- and α,γ-elimination (cleavage of both the CγS and CβS bonds), while Ser77Glu abolishes activity toward cystathionine (PMID:30036991, PMID:36883335). Crystal structures of native TgCGL and the TgCGL-N360S variant bound to cystathionine, cysteine, and the inhibitor d,l-propargylglycine define the molecular basis of substrate binding, with propargylglycine acting as an irreversible active-site inhibitor and cysteine also inhibiting the enzyme (PMID:36883335).

1. 2018 High

   Establishing what reaction TgCGL catalyzes and why its specificity differs from the human enzyme addressed whether the parasite enzyme is a selective drug target; the work showed it is a near-exclusive cystathionine α,γ-lyase governed by two divergent active-site residues.

   Evidence Recombinant enzyme kinetics with structure-guided homology modelling and active-site mutagenesis (N360S, S77E variants)

   PMID:30036991

   Open questions at the time

   • Specificity determinants inferred from homology model rather than experimental structure at this stage
   • No in vivo demonstration of enzyme function in the parasite
2. 2020 High

   Whether TgCGL operates within a complete cysteine-biosynthesis route was unresolved; reconstituting the two-step pathway showed TgCBS and TgCGL together form an intact reverse transsulfuration pathway, placing TgCGL as the cystathionine-consuming terminal enzyme.

   Evidence In vitro reconstitution with recombinant TgCBS and TgCGL, demonstrating TgCBS-generated cystathionine is converted to cysteine by TgCGL

   PMID:32887901

   Open questions at the time

   • Pathway shown in vitro; flux and essentiality in living parasites not established
   • Regulation of the pathway not addressed
3. 2023 High

   The molecular basis of substrate binding and inhibition was undefined; crystal structures of native and N360S TgCGL with bound cystathionine, cysteine, and propargylglycine resolved the catalytic cavity, defined the irreversible inhibition mechanism, and confirmed that N360S broadens bond cleavage to both CγS and CβS.

   Evidence X-ray crystallography of native TgCGL and TgCGL-N360S co-crystallized with substrates and the inhibitor d,l-propargylglycine

   PMID:36883335

   Open questions at the time

   • No structure-based inhibitor optimization or selectivity validation against human CGL reported
   • Cellular consequence of enzyme inhibition in the parasite not tested

• Whether TgCGL and the reverse transsulfuration pathway are essential for T. gondii viability or virulence in vivo, and whether active-site inhibitors translate into antiparasitic efficacy, remains uncharacterized in the available corpus.
• No genetic knockout or in vivo essentiality data
• No demonstration of antiparasitic activity for propargylglycine or other inhibitors