The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan)

Abstract

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of β-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked β-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

Scheme 1. Relationship between Pentyl Pantothenamide (N5-Pan) and Regulation of Pantothenate Biosynthesis

(a) N5-Pan 1 is metabolized by PanK, CoaD, and CoaE to generate ethyl dethiacoenzyme A (EtdtCoA, 2). (b) Pathway from l-aspartate to coenzyme A. β-Alanine 4 is produced by decarboxylation of l-aspartate 3 by aspartate α-decarboxylase (PanD). β-Alanine then forms pantothenate 5, which is subsequently metabolized by PanK, CoaB, CoaC, CoaD, and CoaE to form coenzyme A 3. PanD is produced as a zymogen (proPanD) that is activated by the PanZ·RCoA complex but is also inhibited by the same complex. RCoA = AcCoA or CoA.

Figure 1

(a) Crystal structure of the PanZ·AcCoA–ADC complex at 1.16 Å resolution that reveals the pyruvoyl cofactor of activated ADC is present as a ketone hydrate in the complex. The 2F_o – F_c electron density is shown contoured at one rmsd as gray mesh. ADC carbons are colored yellow and PanZ carbons cyan (limited to residue N45, bottom); this figure was generated using PyMol. (b) Hydrogen bonding interactions in the vicinity of the pyruvoyl group in the apo state (PDB entry 1aw8(10b)). The pyruvoyl keto group forms hydrogen bonds to solvent molecules. Residue K9 forms hydrogen bonds to Y58, H11, and the carboxylate of G24 (formed as a result of the activation reaction). (c) Hydrogen bonding interactions in the PanZ·AcCoA–ADC complex (PDB entry 5ls7, this work). A methyl-ketone hydrate form of the pyruvoyl cofactor is stabilized by hydrogen bonds to G24, T57, and N72. Binding of PanZ to the surface of PanD leads to formation of a hydrogen bond between N45 of PanZ and the backbone carbonyl of E23, distorting the hydrogen bonding network in the active site and displacing H11 from binding to K9.

Figure 2

Regulation of ADC is due to CoA-dependent interaction of PanZ and ADC. (a) Overexpression of PanZ is sufficient to generate β-alanine auxotrophic bacteria. Growth of MG1655 is inhibited on M9 arabinose medium in the absence of β-alanine. In contrast, growth of strain SN218, in which the panD locus is replaced with that from B. subtilis, is not perturbed by PanZ overexpression. (b and c) Screening of mutations in panZ and panD to identify site-directed mutants that can relieve inhibition but maintain growth. (b) A K119A mutation in the chromosomal panD locus leads to loss of growth suppression. (c) Overexpression of panZ(R73A) does not inhibit cell growth. (d) Analysis of protein complex formation by SEC. The WT PanD–PanZ complex elutes as a heterooctamer (light gray line), whereas the PanD(K119A)–PanZ complex elutes as a mixture of the heterooctamer, PanD tetramer, and PanZ monomer (dashed line). The PanD–PanZ(R73A) mixture elutes as independent tetramer and monomer components (dark gray line). (e and f) Calorimetric analysis of interaction of PanZ(R73A) and AcCoA. (e) The loss of Arg73 from the AcCoA binding site reduces the affinity of the protein for AcCoA by ∼250-fold. (f) Titration of PanZ(R73A) into PanD(S25A) in the presence of high concentrations of AcCoA (1 mM) indicates that the proteins interact at physiological concentrations of AcCoA.

Figure 3

Testing of E. coli strains for growth inhibition by pentyl pantothenamide. (a) Titration of AcCoA against purified PanZ by ITC reveals substoichiometric but tight binding of AcCoA, due to co-purification of CoA with PanZ. (b) Binding of EtdtCoA to PanZ is indistinguishable from that of AcCoA [note the sloping baseline due to residual salt in the metabolite preparation (see Figure S5 for details of global fitting)]. (c) Growth of WT E. coli (MG1655) and a ΔpanD::BspanD strain (SN218) on solid M9 agar medium supplemented with pentyl pantothenamide and β-alanine (0.5 mM). (d and e) Growth curves for MG1655 and SN218 (ΔpanD::BspanD), respectively, in liquid culture. Residual growth is observed even at inhibitory concentrations of the compound. (Inset − concentration of N5-Pan in μg mL⁻¹).