AAT

PF3D7_0204500 (AspAT) is a pyridoxal-5'-phosphate-dependent aspartate aminotransferase of P. falciparum that interconverts aspartate and α-ketoglutarate with oxaloacetate and glutamate and supplies aspartate for downstream biosynthesis (PMID:11443076, PMID:21209090). The recombinant enzyme catalyzes aspartate–ketoglutarate, tyrosine–ketoglutarate, and amino acid–ketomethiobutyrate transaminations, and it performs the final transamination step of methionine regeneration from methylthioadenosine, albeit with ketomethiobutyrate turned over much more slowly than aspartate–ketoglutarate; canaline acts as an uncompetitive inhibitor (Ki 27 µM) (PMID:11443076). Metabolically, AspAT operates downstream of the mitochondrial electron transport chain, converting fumarate-derived carbons (via malate and oxaloacetate) into aspartate that feeds nucleic acid and protein synthesis, a route abolished by the ETC inhibitor atovaquone (PMID:21209090). The 2.8 Å crystal structure shows the enzyme is a cytosolic homodimer adopting the canonical AspAT fold but with a divergent N-terminal region; deletion of these N-terminal residues abolishes catalytic activity, and a peptide of the 13 divergent N-terminal residues inhibits the parasite enzyme in vitro and in lysates without affecting human cytosolic AspAT, marking this region as a basis for parasite-selective inhibition (PMID:21087616). Notably, the P. falciparum enzyme lacks the residue G197 that is invariant across the Ia aminotransferase subfamily (PMID:11443076).

1. 2001 High

   Establishing the catalytic identity and substrate range of AspAT was the first step in defining its metabolic role, showing it is a broad-specificity aminotransferase that also completes methionine regeneration.

   Evidence recombinant expression with multi-substrate in vitro kinetic assays and inhibitor (canaline) characterization

   PMID:11443076

   Open questions at the time

   • Relative in vivo importance of the methionine-regeneration versus aspartate-synthesis activities not resolved
   • Physiological significance of the missing G197 residue for catalysis not tested
2. 2010 Medium

   Determining the enzyme was amenable to crystallization enabled subsequent structural analysis.

   Evidence recombinant purification, crystallization, and 2.8 Å X-ray diffraction data collection

   PMID:20383010

   Open questions at the time

   • No structural interpretation yet at this stage
   • Superseded by the full structure determination
3. 2010 High

   The crystal structure and N-terminal mutagenesis revealed that a divergent N-terminal region is essential for catalysis and offers a parasite-selective inhibition handle, and localized the enzyme to the cytosol.

   Evidence X-ray structure of the homodimer at 2.8 Å, N-terminal deletion, N-terminal peptide inhibition in vitro and in lysates, subcellular localization

   PMID:21087616

   Open questions at the time

   • Mechanism by which the N-terminal peptide inhibits activity not resolved
   • No inhibitor developed from the N-terminal region against live parasites
4. 2011 High

   Isotope tracing placed AspAT in a defined electron-transport-chain-dependent pathway that routes fumarate carbon into aspartate for nucleic acid and protein biosynthesis, giving the enzyme a concrete metabolic role in intraerythrocytic parasites.

   Evidence [2,3-14C] and [2,3-13C]fumarate labeling with NMR metabolite tracing and atovaquone inhibition in free parasites

   PMID:21209090

   Open questions at the time

   • Genetic essentiality of AspAT for this pathway not tested
   • Direct enzymatic contribution inferred pharmacologically rather than by AspAT-specific perturbation

• Whether AspAT is genetically essential and whether its N-terminal region can be exploited for a parasite-selective antimalarial in vivo remains open.
• No genetic knockout/knockdown of AspAT in the corpus
• No in vivo antimalarial validation of N-terminal-targeting inhibitors