CBS

CBS in Toxoplasma gondii (TgCBS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme of the reverse transsulfuration pathway that catalyzes cystathionine biosynthesis and also generates hydrogen sulfide (H2S) (PMID:32887901). In β-replacement reactions it uses either serine or O-acetylserine together with homocysteine to form cystathionine via an aminoacrylate intermediate, and it preferentially produces H2S by condensing cysteine and homocysteine (PMID:32887901). Its substrate preference between serine and O-acetylserine is set by active-site residues S84, Y160, and Y246, with Y246 dominant in stabilizing the serine leaving group; the S84A/Y160F/Y246V triple mutant converts the enzyme into an O-acetylserine-exclusive (OCBS-like) form and lowers the pH optimum (PMID:37163386). Structurally, TgCBS lacks the N-terminal heme-binding domain of human CBS and assembles into a unique basket-like dimer while retaining two C-terminal CBS (Bateman module) domains (PMID:32887901, PMID:34194677). Unlike human CBS, TgCBS is not stimulated by S-adenosylmethionine and its Bateman module does not exert autoinhibitory control — removal by deletion, proteolysis, or thermal denaturation leaves activity, oligomerization, and stability unchanged — defining a distinct regulatory mechanism (PMID:32887901, PMID:35897745).

1. 2020 High

   Establishing that T. gondii encodes a functional CBS answered whether the parasite runs a reverse transsulfuration pathway and revealed it uses a heme-independent, SAM-insensitive enzyme with dual cystathionine- and H2S-producing activities.

   Evidence biochemical characterization of recombinant TgCBS with multiple substrates, intermediate trapping, and SAM stimulation assays

   PMID:32887901

   Open questions at the time

   • In vivo role in parasite physiology and whether both activities matter for infection not tested
   • Kinetic contributions of the two C-terminal CBS domains not resolved
2. 2021 High

   The crystal structure defined the architecture underlying TgCBS's heme independence, showing a basket-like dimer lacking the N-terminal heme domain and resembling the pathogenic human D444N conformation.

   Evidence X-ray crystallography with bound reaction intermediates, molecular dynamics, and activity assays of a catalytically intact deletion construct (Δ466-491)

   PMID:34194677

   Open questions at the time

   • Functional consequence of the D444N-like arrangement for catalysis not directly probed
   • Structure of the full Bateman module region not resolved
3. 2022 High

   Testing whether the C-terminal Bateman module autoinhibits TgCBS as it does in human CBS established a novel regulatory mode in which the module is dispensable for activity control.

   Evidence deletion mutagenesis, limited proteolysis, and thermal denaturation, each assessed for activity, oligomerization, and thermal stability

   PMID:35897745

   Open questions at the time

   • Actual physiological function of the retained Bateman module unknown
   • Whether any ligand regulates TgCBS through this module not determined
4. 2023 High

   Identifying S84, Y160, and Y246 as determinants of serine/O-acetylserine preference explained the molecular basis of TgCBS's dual-substrate capability and how it can be reprogrammed toward OCBS-like behavior.

   Evidence single and triple site-directed mutagenesis with activity assays, aminoacrylate trapping, and protein-aminoacrylate complex structures

   PMID:37163386

   Open questions at the time

   • Whether O-acetylserine is a physiologically relevant substrate in vivo not established
   • Relative flux through cystathionine versus H2S production in the cell not quantified

• The in vivo function of TgCBS in T. gondii metabolism, growth, and pathogenesis, and whether its distinct regulation is exploitable as a drug target, remain open.
• No genetic knockout or conditional depletion characterized
• No demonstrated contribution to virulence or metabolic phenotype in the parasite