Volume 10, Issue 12, Pages 1293-1302 (December 2003) Assembly of the Covalent Linkage between Lipoic Acid and Its Cognate Enzymes  Xin Zhao, J.Richard Miller, Yanfang Jiang, Michael A Marletta, John E Cronan  Chemistry & Biology  Volume 10, Issue 12, Pages 1293-1302 (December 2003) DOI: 10.1016/j.chembiol.2003.11.016

Figure 1 Growth of lip Mutants on Minimal Medium The strains were streaked onto plates of glucose minimal E medium supplemented as shown above the plates in the pattern given at the left of each row of plates. The strains were the wild-type (WT) strain JK1, KER176 (lipA), KER184 (lipB), TM134 (lplA), TM131 (lplA lipA), and TM136 (lplA lipB). The supplements to the medium were added to final concentrations of acetate, 5 mM; succinate, 5 mM; lipoic acid, 5 ng/ml (∼25 nM); and octanoic acid, 50 μM. The plates were incubated 24 hr at 37 oC. Chemistry & Biology 2003 10, 1293-1302DOI: (10.1016/j.chembiol.2003.11.016)

Figure 2 The Proposed Pathway of Lipoic Acid Synthesis The rounded rectangle is an E. coli cell, and the ovals are the E2 domains of the 2-oxoacid dehydrogenases. The carboxyl group of the octanoate and lipoate moieties are in amide linkage (data not shown) to the ϵ-amino group of a specific domain lysine residue. Chemistry & Biology 2003 10, 1293-1302DOI: (10.1016/j.chembiol.2003.11.016)

Figure 3 Nondenaturing Gel Electrophoretic Characterization of Various HPLC Fractions of the E2-Domain Purification CE, AP, and AS denote the crude extract, acid treatment pellet, and acid treatment supernatant, respectively, whereas apo denotes an apo-E2 domain standard. Selected fractions from the HPLC-ion exchange chromatography column are shown. All of the HPLC fractions of the upper gel are the apo form, whereas all of the fractions in the lower gel are modified species. The loss of the positive charge of the modified lysine ϵ-amino group accounts for the differences in gel mobility and column elution of the apo and modified species. Chemistry & Biology 2003 10, 1293-1302DOI: (10.1016/j.chembiol.2003.11.016)

Figure 4 Electrospray Mass Spectra of Purified Modified E2-Domain Fractions Strain ZX137A having the genotype lipA lipB fadE (λ) was grown in the presence of octanoic-d15 acid. The cells were then harvested, washed free of octanoic-d15 acid, and resuspended in growth medium lacking octanoate acid (A–F) or medium containing unlabeled octanoate (G and H). After 1 hr of growth, the culture was split in half and LipA function was restored to one half. Modified E2 domains were purified as described in Experimental Procedures. H15-C8 and D15-C8 denote the hydrogen and deuterium (octanoic-d15 acid) forms of octanoyl-E2 domain, respectively, whereas H15-Lip and D13-Lip denote the hydrogen and deuterium (lipoic-d13 acid) forms of the lipoyl-E2 domain, respectively. The masses of the major peaks are given in the panels. In each pair of panels, the same fractions from the two HPLC separations (Figure 3) are compared for the LipA-containing and control cultures. (A), (C), and (E) are fractions from the parallel control culture in which LipA function was not restored. (B), (D), and (F) are fractions from the culture in which LipA function was restored by transduction with the lipA cosmid, pZX130. (A) and (B) are HPLC fraction 29, (C) and (D) are fraction 27, and (E) and (F) are fraction 31. In (G) and (H), unlabeled octanoic acid was added following removal of octanoic-d15 acid, and the protein fractions analyzed were a pool of HPLC fractions 24–27. Note the increased levels of the H15-C8 species compared to (A)–(F) and the appearance of detectable levels of the H13-Lip species. The unlabeled peaks are ammonia (or occasionally sodium or potassium) adducts. Chemistry & Biology 2003 10, 1293-1302DOI: (10.1016/j.chembiol.2003.11.016)

Figure 5 Consumption of Tritiated SAM in the Presence of Reduced LipA and Octanoylated-E2 Domain The assay products were separated by HPLC and and detected with an in-line flow scintillation counter (see Experimental Procedures). The upper chromatogram shows the products of a reaction in which only LipA was omitted. The lower chromatogram shows the same reaction components in the presence of reduced LipA. See Table 1 for the assay protocol and components. Chemistry & Biology 2003 10, 1293-1302DOI: (10.1016/j.chembiol.2003.11.016)