Fig 1.
Targeting eFA salvage re-sensitizes bacterial pathogens to killing by cerulenin-included FAS II inhibitors.
A. eFA salvage coupled with FAS II pathway contributes to membrane phospholipid synthesis. Three mechanisms for eFA scavenging were included here, namely (i) FadD acyl-CoA ligase [29]; (ii) FakA/B system composed of the FakA kinase component and the FakB fatty acid-binding subunit [25,28]; (iii) Acyl-ACP synthetase, AasS [47,48]. Cerulenin denoted the FabF inhibitor targeting a FAS II pathway. B. The combination of PlsB/Y-PlsC and PlsX/Y-PlsC represented two alternative routes for the synthesis of membrane phospholipids. The pathway begins with G3P as a recipient, and extends using different primer substrates (acyl-CoA/acyl-ACP for PlsB/PlsC vs acyl-Pi for PlsY/X, and acyl-ACP for PlsC). Abbreviations: C10-AMS, 5’-O-(N-decanylsulfamoyl) adenosine; FadD, Acyl-CoA ligase; FakA/B, Fatty acid kinase A in complex with fatty acid-biding subunit B; AasS, acyl-ACP synthetase; AccABCD, Acetyl-CoA carboxylase composed of four subunits (namely (i) AccA, α-subunit of carboxyltransferase; (ii) AccB, biotin carboxyl carrier protein (BCCP); (iii) AccC, biotin carboxylase (BC); and (iv) AccD, β-subunit of carboxyltransferase); FAS II, Type II fatty acid synthesis pathway; FabI, Enoyl-ACP reductase; FabF, β-ketoacyl-ACP synthase II; FabG, Ketoacyl-ACP reductase; FabZ, 3-hydroxyacyl-ACP dehydratase; G3P, Glycerol-3-phosphate; LPA, Lyso-phosphatidic acid; PA, Phosphatidic acid; acyl-Pi, acyl phosphate; PlsB, G3P acyltransferase; PlsY, Acyl-Pi-dependent G3P acyltransferase; PlsX, Phosphate: acyl-ACP transacylase; PlsC, LPA acyltransferase; G-, Gram-negative bacterium; G+, Gram-positive bacterium.
Fig 2.
AasS activity with C10 fatty acid substrate is abolished by the C10-AMS inhibitor.
Chemical structures of an intermediate C10-adenylate (A) and the inhibitor C10-AMS (B). C. The principle for ligation of decanoic acid (C10) by AasS with holo-ACP to generate C10-ACP, and its inhibitory mechanism by C10-AMS. Qualitative analysis (D) and relative quantitation curve (E) of C10-ACP conversion from C10 substrate in a dose-dependent manner. In the reaction system of AasS (3 nM), unlike the ACP acceptor protein that was added at the constant level of 3 μg (panels D&E), the level of C10 fatty acid varied markedly (6, 12, 24,…, to 240 μM). F. The AasS-catalyzed production of C10-ACP in vitro, is abolished by C10-AMS inhibitor in a dose-dependent pattern. The conformationally-sensitive gel of 0.5 M urea/17.5% PAGE (pH9.5) was utilized to separate C10-ACP from its acceptor holo-ACP (panels D&F). G. Semi-quantitative assays for inhibition of AasS catalysis by C10-AMS compound. The determined Ki of C10-AMS vs AasS is 0.62 μΜ. Except for the C10 substrate that was fixed at 80 μM, AasS reaction was established identically as described in Fig 2D and 2E. It was noted that the ratio of C10-AMS inhibitor to AasS varied dramatically (ranging from 0:1, 250:1, 500:1, 1000:1, 2000:1, to 4000:1). Designations: ACP, acyl carrier protein; ATP, adenosine triphosphate; PPi, pyrophosphate.
Fig 3.
ITC analysis for binding of C10-AMS inhibitor to AasS enzyme.
A. Scheme for an intermediate of C10-AMP generated from the C10 fatty acid substrate via the ‘first-half’ reaction of AasS activation. B. Use of ITC to measure an interaction between AasS enzyme and its C10-AMP intermediate. C. ITC-based assay for AasS specifically bound by the C10-AMS inhibitor. Designations: ITC, isothermal titration calorimetry; N, stoichiometry; Kd, dissociation constant; DP, differential power; ΔH, enthalpy.
Fig 4.
Integrated evidence for the inhibition of AasS action with E-C7 fatty acid by C10-AMS compound.
A. Schematic diagram of AasS-catalyzed ligation of E-C7 with holo-ACP recipient to produce E-C7-ACP. B. Use of conformationally-sensitive PAGE to visualize the fact that the C10-AMS inhibitor displays dose-dependent inhibition on the conversion of E-C7-ACP ester from its acceptor holo-ACP. A representative photograph of three independent trials was given. C. Relatively-quantitative analysis (for the inhibitory efficacy of C10-AMS on AasS-catalyzed E-C7-ACP production. As for AasS reaction (panel B), unlike the three components that are at constant levels, namely (i) AasS (31.25 nM), (ii) E-C7 (600 μM), and (iii) ACP (3 μg), C10-AMS inhibitor was supplemented at varied ratio in relative to the AasS enzyme (ranging from 0:1, 25:1, 50:1, 100:1, 200:1, 300:1, 400:1, to 500:1). The ImageJ software was applied to measure the relative percentage (%) of E-C7 fatty acylation in each AasS reaction. The resultant graphs were plotted from three independent experiments, in which the output was expressed as means ± SD (standard deviations). D. Schematic diagram for C10-AMS inhibition of AasS-based bypass of biotin requirement by the E. coli ΔbioC biotin auxotroph on the non-permissive condition. E. Altered viability of AasS-expressing E. coli ΔbioC strain suggested the in vivo inhibition of C10-AMS in a dose-dependent manner. Using M9 defined medium with E-C7 as sole carbon source (displayed in panel on left hand), biotin bypass assays were performed with the ΔbioC strain that produces AasS enzyme carried by a plasmid. Log-phase cultures in a series of 10-fold dilution (shown in panel on right hand), were spotted on the x-gal-containing M9 agar plates supplemented with C10-AMS inhibitor at varied levels (0, 50, 100, to 500 μM). The addition of x-gal substrate enabled the occurrence of blue colonies for better photographing, and viable colonies were highlighted with yellow arrows.
Fig 5.
Structural characterization of AasS complexed with its inhibitor C10-AMS.
A. Linear presentation of full-length AasS enzyme composed of two domains. The large domain AasS_N (residues 1–424) is connected by a short linker (residues 425–430) with the compact small domain, AasS_C (residues 431–533). Ribbon illustration (B) and surface structure (C) of AasS hexamer liganded with C10-AMS inhibitor. The AasS hexamer (130 x 140 x 65 Å) essentially behaves as a trimer of dimers, in which the subunit of monomer is numbered from I, II, …, to VI. The dimer interface was highlighted with a red arrow, and the trimer interface was indicated with a blue arrow. The AasS top view (130 x 140 Å, in upper panel) was rotated 90° counter-clockwise, giving its front view (65 x 140 Å, in bottom panel). The N-terminal domain of AasS was colored blue for AasS_N (or magenta for AasS’_N), and the C-terminal domain of AasS was displayed in powder blue for AasS_C (or light pink for AasS’_C).
Fig 6.
The binding of C10-AMS inhibitor promotes a markedly-conformational rearrangement of AasS enzyme.
A-B. Ribbon and surface representation of AasS protomer in an apo-form (PDB: 8HSY). As for apo AasS, its AasS_C domain colored pink, appears in an ‘open’ orientation. C-D. Cartoon and surface structure of AasS protomer in complex with its product M-C7-ACP (PDB: 8I8D). In this complex, the AasS_C part colored yellow, displays a ‘closed’ orientation. The ACP partner is colored lime-green. Ribbon structure (E) and surface illustration (F) of an AasS enzyme liganded with a molecule of C10-AMP intermediate per protomer (PDB: 8JYU). The occupancy of C10-AMP intermediate renders the AasS_C domain (colored hot-pink) in an ‘open’ orientation. The C10-AMP molecule is given in a stick representation, whose carbon atoms are colored cyan. G-H. Cartoon and surface characterization of C10-AMS inhibitor-liganded AasS protomer (PDB: 8JYL). The effective binding of C10-AMS inhibitor leads to a ‘closed’ orientation of AasS_C domain (in gold) converted from its “open” orientation. Like a C10-AMP molecule, the C10-AMS inhibitor is also given in a stick representation, of which carbon atoms are colored marine. Designations: AasS_C, C-terminal domain of AasS; AasS_N, N-terminal domain of AasS.
Table 1.
Cryo-EM Collection, refinement and validation statistics of AasS enzyme liganded with C10-AMS inhibitor or C10-AMP intermediate.
Fig 7.
Parallels in binding of AasS to the C10-AMP adenylate intermediate and the C10-AMS inhibitor.
Structural visualization for AasS binding an intermediate C10-AMP adenylate (A) and its inhibitor C10-AMS (B). Mg2+ atoms are displayed as spheres and residues closer to the position of ligands are shown in sticks form. New residues were seen as a result of the C-terminus rearrangement, as the pink sticks showed, with K432 being especially important. C. The three mutants of AasS (D411A, R426A, and K432A) are inactive with the C10 fatty acid substrate in vitro. Both AasS enzymatic reaction and conformationally-sensitive gel separation were conducted as described in Fig 2D and 2F. D. In vivo evidence that the three single mutants of AasS (D411A, R426A, and K432A) lose the ability to bypass the physiological requirement of biotin for the bioC isogenic mutant of E. coli on the non-permissive condition. The bacterial viability on M9 defined agar medium was determined as performed in Fig 4E. E. ITC experiments revealed that WT of AasS binds to decanoic acid substrate, whereas the AasS (D411A) mutant does not. The ITC assays were carried out as we conducted in Fig 3B.
Fig 8.
Schematic diagram for inhibition of AasS by the C10-AMS inhibitor.
A. The cut-away view of a C10-AMP intermediate-loaded cavity of AasS enzyme (PDB: 8JYU). The C10-AMP molecule is shown in a stick representation and carbon atoms are colored in cyan. B. The cut-away view of AasS enzyme revealed that C10-AMS inhibitor occupies the C10-AMP intermediate-binding cavity (PDB: 8JYL). Like a C10-AMP molecule, the C10-AMS inhibitor is also given in a stick representation, of which carbon atoms are colored in yellow. C. The cut-away view of ACP cargo and released AMP in the substrate cavity of AasS/M-C7-ACP complex (PDB: 8I8D). The carbon atoms of Ppan arm, M-C7 and AMP are colored in pink, green, and magenta respectively. Chemical structures of C10-AMP intermediate (D) and C10-AMS inhibitor (E). It was given in ball-and-stick models. Red balls denoted oxygen atoms, blue balls referred to nitrogen atoms, and orange balls indicated phosphate atoms. Except for a sulfamoyl moiety that replaces a phosphate group, the C10-AMS inhibitor resembles the C10-AMP intermediate. F. Cartoon representation for open conformation of a two-subunit enzyme AasS, of which a large domain AasS_N contains a V-shape cavity for sequential loading of three distinct substrates (S1 to S3). Namely, they included decanoic acid (S1), ATP (S2), and ACP (S3). G. A working model for AasS in open conformation adapted to sequential binding its three unique substrates (S1 to S3). H. Scheme for C10-AMS inhibitor-induced closed formation of AasS that blocks the entry of three substrates (S1 to S3).