Fig. 3: Glutaryl–lipoyl thioesterase activity of ABHD11.
From: Lipoyl deglutarylation by ABHD11 regulates mitochondrial and T cell metabolism
a, Schematic of assay to detect removal of glutaryl moieties from synthetic OGDHc-E2 peptides by ABHD11-Flag. Thioesterase activity is measured by the release of free thiols, which react with Ellman’s reagent (DTNB) and can be detected via absorbance at 412 nm. Alternatively, glutarate release is detected using LC–MS. b,c, Thioesterase activity of ABHD11-Flag (100 nM) on unmodified, Lp or Lpglu OGDHc-E2 peptides (100 µM) was determined via DTNB assay. Mean ± s.d.; n = 3; one-way ANOVA and Dunnett’s post hoc test. d,e, Thioesterase activity of ABHD11-Flag (100 nM) on OGDHc-E2 Lpglu peptide (100 µM) in the presence or absence of ML226 (2.5 µM) was determined via DTNB assay. Mean ± s.d., n = 4 independent experiments, one-way ANOVA and Tukey’s post hoc test. f, In silico modeling of ABHD11 to a marine α/β-hydrolase fold esterase (RCSB PDB ID: 7c4d.1; 2.03 Å) with lipoate moiety. Catalytic S141 and H296 indicated. Ribbon (left) and surface charge (right) models shown. g,h, Glutarate release from OGDHc-E2 Lpglu (g) or Kglu (h) peptides (100 µM) incubated with ABHD11-Flag (100 nM) in the presence or absence of ML226 (2.5 µM) was determined using LC–MS. Mean ± s.d., n = 3 independent experiments, one-way ANOVA and Dunnett’s post hoc test. RCSB PDB, Research Collaboratory for Structural Bioinformatics Protein Data Bank.
