An international research team led by Nagasaki University has elucidated, at the atomic level, the three-dimensional structure and reaction mechanism of the key energy-metabolism enzyme acetate: succinate CoA-transferase (ASCT) present in the mitochondria of Trypanosoma brucei (T. brucei), the protozoan parasite that causes African sleeping sickness (Human African trypanosomiasis).

In this study, the researchers conducted a detailed comparison of ASCT with the related human enzyme, succinyl-CoA transferase (SCOT). Through site-directed mutagenesis analysis, they discovered for the first time in the world that a difference in just a single amino acid residue determines the enzyme’s reaction specificity. Substitution of this amino acid was shown to increase over 4000-fold the SCOT activity that is not originally present in ASCT.

These findings not only reveal the molecular basis of parasite-specific energy metabolism but also provide an important clue for the development of selective antiparasitic drugs that do not affect human metabolism.

Abstract

Trypanosomatids are protozoan parasites that remain a global health challenge due to the limited efficacy, safety, and durability of current treatments. Acetate: succinate CoA transferase (ASCT), together with succinyl-CoA synthase (SCS), forms the ASCT/SCS cycle that fuels ATP production and generates acetate, a central metabolic intermediate essential for mitochondrial pathways in these parasites. Although Trypanosoma brucei ASCT (TbASCT) shares 52% amino acid identity with mammalian succinyl-CoA:3-ketoacid CoA transferase (mSCOT), the latter catalyzes a rate-limiting step of ketone body catabolism. Because ASCT and SCOT perform distinct reactions, understanding their mechanistic divergence is crucial for identifying parasite-specific vulnerabilities and advancing selective drug discovery. Here, we report crystal structures of TbASCT bound to all substrates and products, revealing the molecular basis of substrate recognition and catalysis. In solution, TbASCT and mSCOT are homotetrameric and homodimeric, respectively. Despite similar monomer fold, the substrate-binding sites and catalytic mechanisms by which these two enzymes mediate different reactions remain unknown. Site-directed mutagenesis demonstrated that residues Arg162, Leu377, and Asp62 govern tetramer assembly, CoA binding, and acquisition of SCOT activity, respectively. Mutation of Leu377 abolished ASCT activity, while Arg162 mutant produced ASCT-active dimers with a 10-fold increase in SCOT/ASCT activity ratio. Notably, Asp62 mutants exhibited more than 4000-fold increase in this ratio, representing gain-of-function SCOT activity, a reaction absent in TbASCT. These mechanistic insights define the structural determinants that separate ASCT from SCOT function and illuminate opportunities to selective inhibit ASCT without disrupting host SCOT, thereby informing the development of trypanosomatid-targeted therapeutics.

Journal Information

Journal: Protein Science

Title: Modulation of succinyl-CoA:3-ketoacid CoA transferase activity by a single amino acid residue in acetate:succinate CoA transferase from Trypanosoma brucei, the causative agent of African sleeping sickness

Authors: Kota Mochizuki, Daniel Ken Inaoka, Keisuke Fukuda, Hana Kurasawa, Kenji Iyoda, Uta Nakai, Shigeharu Harada, Emmanuel O. Balogun, Frédéric Bringaud, Michael Boshart, Takaya Sakura, Kenji Hirayama, Kiyoshi Kita, Tomoo Shiba

DOI: 10.1002/pro.70463

For more details, please refer to the full article published in Protein Science.