GAPDH1

Toxoplasma gondii GAPDH1 is an essential multifunctional glycolytic enzyme required for intracellular replication, though its glycolytic catalytic activity per se is not strictly essential (PMID:27859784). A 2.25 Å crystal structure resolves GAPDH1 as a quaternary holoenzyme, providing the framework for its structure-function analysis (PMID:27859784). Enzymatic activity is regulated by S-loop phosphorylation, a post-translational modification acting through an S-loop required for cofactor binding, allosteric activation, and oligomerization (PMID:27859784). Independently of its catalytic and phosphorylation state, GAPDH1 translocates to the cortical membrane skeleton upon egress via an N-terminal, palmitoylation-dependent mechanism, establishing that its glycolytic and cortical-association functions are decoupled post-translational events (PMID:27859784). Beyond these findings from a single structural and functional study, no further mechanistic detail has been characterized in the available corpus.

1. 2016 High

   Resolving whether GAPDH1's essentiality derives from glycolysis established that the enzyme is required for intracellular replication independent of its strict glycolytic role.

   Evidence inducible knockdown plus catalytic-site mutagenesis with intracellular replication readout in T. gondii

   PMID:27859784

   Open questions at the time

   • the non-glycolytic function accounting for essentiality is not defined
   • no identification of downstream metabolic or structural pathways affected by knockdown
2. 2016 High

   The holoenzyme crystal structure provided the atomic framework needed to interpret how modifications and mutations alter GAPDH1 function.

   Evidence X-ray crystallography at 2.25 Å resolution of the T. gondii GAPDH1 quaternary complex

   PMID:27859784

   Open questions at the time

   • no structural model of the phosphorylated versus unphosphorylated S-loop state
   • no structure of the palmitoylated N-terminus or membrane-associated form
3. 2016 High

   Identifying S-loop phosphorylation defined a post-translational switch coupling cofactor binding, allosteric activation, and oligomerization to enzymatic output.

   Evidence phosphosite identification with structural analysis, enzymatic activity assays, and S-loop mutagenesis in T. gondii

   PMID:27859784

   Open questions at the time

   • the kinase responsible for S-loop phosphorylation is not identified
   • physiological conditions triggering phosphorylation are undefined
4. 2016 High

   Live imaging of egress-triggered relocalization established a second, catalytically independent function: N-terminal palmitoylation-dependent recruitment to the cortical membrane skeleton.

   Evidence live imaging of GAPDH1 during egress, N-terminal deletion/mutagenesis, and palmitoylation inhibition in T. gondii

   PMID:27859784

   Open questions at the time

   • the palmitoyltransferase and modified residue are not identified
   • the functional consequence of cortical association during egress is not defined
   • the signal linking egress to translocation is unknown

• The non-glycolytic function underlying GAPDH1 essentiality and the role of its egress-triggered cortical association remain unresolved.
• no mechanistic link between cortical localization and replication essentiality
• no identification of the regulatory kinase or palmitoyltransferase
• no characterization across T. gondii strains