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)

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.


Figures S1-S7 S3
Experimental methods S10 Analytical data for pentyl pantothenamide and ethyldethiacoenzyme A S14 S3 Figure S1 Overlay of structure of ADC(T57V)-PanZ complex (green and cyan) and ADC(WT)-PanZ complex (yellow and white). Figure S2 a Fo-Fc difference electron density (green mesh) contoured at 3 rmsd and 2Fo-Fc electron density (grey mesh) contoured at 1 rmsd showing the presence of additional density in the region of the pyruvoyl group (green mesh). b final electron density, contoured as in (a) with a ketone hydrate modeled.

Figure S3 Size exclusion chromatography analysis of complex formation by PanD and
PanZ. a&b SEC analysis of PanD (a black), PanD(K119A) (a red), PanZ (b black) and PanZ(R73A) (b red). All proteins elute as single species; analysis of peak fractions by SDSpage reveals that PanD electrophoreses as a tetramer if samples are not preboiled. c SEC analysis of WT PanD-PanZ complex. The complex elutes as a single species, Western blotting analysis confirms the presence of PanZ in the complex, trace activated PanD is visible in the boiled fractions. d SEC analysis of PanD(K119A) interaction with PanZ. PanZ chiefly elutes as separate species but a small proportion elutes in a complex with PanD(K119A). e SEC analysis of PanZ(R73A) interaction with PanD. The proteins elute individually.

Figure S4
Relationship of Arg73 to AcCoA binding site in PanZ. Binding site for AcCoA in the PanD(WT)-PanZ.AcCoA crystal structure. Residue Arg73 forms a salt-bridge with residue Glu103 adjacent to the pantothenate moiety of AcCoA, potentially forming both van der Waals interactions and stabilizing the helix which forms the adenine binding pocket. 2Fo-Fc electron density contoured at 1 rmsd for AcCoA, Arg73 and Glu103.

Figure S5
Global fitting of EtdtCoA binding to PanZ by isothermal titration calorimetry. The sample of EtdtCoA contained trace impurities leading to a significant sloping heat of dilution (see red lines S5a). Four independent titrations were globally fitted in SEDPHAT to a single set of thermodynamic parameters but with independent baseline height and slope correction. Substoichiometric binding is due to copurification of the AcCoA with PanZ. a 85 M EtdtCoA titrated into 10 M PanZ, b & c 85 M EtdtCoA titrated into 18 M PanZ, d 85 M EtdtCoA titrated into 20 M PanZ

Figure S6
Estimation of minimum inhibitory concentration for growth of E. coli MG1655 and strains in which the panD locus has been mutated determined by growth in M9 media for 20 h at 37 ºC. Pentyl pantothenamide inhibits growth of wild-type E. coli at 4 mg/mL (Black squares). Mutation of the panD locus to panD(K119A) which reduces the PanD-PanZ interaction affinity is insufficient to generate resistance (red circles) whereas incorporation of the Bacillus subtilis gene at the panD locus (SN218) is sufficient to generate resistance (blue triangles). Data are fitted to a Logistic model for growth inhibition with shared upper and lower limits.  Figure S7 Analysis of growth of E. coli MG1655 inhibited by pentyl pantothenamide. a -Alanine supplementation inhibits growth of E. coli Growth of MG1655 in the presence of increasing concentrations of pentyl pantothenamide and 0.5 mM -alanine indicates that cells grow to a lower final density in the presence of -alanine, suggesting that overproduction of pantothenate under these conditions is detrimental to optimal growth. b Pentyl pantothenamide does not effect growth rate. Growth of MG1655 in increasing concentrations of pentyl pantothenamide (data also shown in figure 3b).The exponential growth rate is broadly unaffected by addition of pentyl pantothenamide, only the plateau position is affected (inset). c Final plateau growth density is a function of seeding density.
Growth of E. coli MG1655 in the presence of 10 g mL -1 pentyl pantothenamide at varied seeding densities indicates a linear relationship between seeding density and the final growth plateau.

Size-exclusion chromatography analysis
Size-exclusion chromatography analysis was carried out as described previously. Following purification, proteins were analysed using an Akta Prime Plus liquid chromatography system at a flow rate of 0.2 mL/min of 50 mM Hepes/KOH, 150 mM KCl, pH 7.6. For proteininteraction analysis 60 M PanD and 15 M PanZ were mixed and incubated at 4 ºC for 60 min prior to analysis. Western blotting against PanZ was carried out using a rabbit polyclonal antibody to PanZ produced under contract by Biogate Co., Japan.