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Formation of a heterooctameric complex between aspartate α-decarboxylase and its cognate activating factor, PanZ, is CoA-dependent.

Monteiro DC, Rugen MD, Shepherd D, Nozaki S, Niki H, Webb ME - Biochem. Biophys. Res. Commun. (2012)

Bottom Line: The existence of a fifth essential protein for pantothenate biosynthesis in some enteric bacteria has recently been reported by Stuecker et al. [10] and Nozaki et al. (in press) [9].This protein, PanZ, catalyses the activation of the PanD zymogen to form ADC and is essential for prototrophic growth.These approaches reveal that the two proteins interact with nanomolar affinity in a CoA-dependent fashion to form a heterooctameric complex.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Leeds, Leeds LS2 9JT, UK.

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Electrospray MS analysis of PanD-T57V and its complex with PanZ-CoA. (A) ESMS spectrum of PanD-T57V tetramer. (B) ESMS spectrum of PanD-T57V in the presence of PanZ-CoA reveals a mixture of species corresponding to a tetramer of PanD-T57V bound to either 3 or 4 equivalents of PanZ-CoA. (C) Simulation of peak positions for PanD–PanZ complexes matches observed peak positions.
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f0010: Electrospray MS analysis of PanD-T57V and its complex with PanZ-CoA. (A) ESMS spectrum of PanD-T57V tetramer. (B) ESMS spectrum of PanD-T57V in the presence of PanZ-CoA reveals a mixture of species corresponding to a tetramer of PanD-T57V bound to either 3 or 4 equivalents of PanZ-CoA. (C) Simulation of peak positions for PanD–PanZ complexes matches observed peak positions.

Mentions: Cort et al. (PDB: 2k5t) noted that after overexpression in E. coli, PanZ is purified as a mixture of free protein and ligand-bound to coenzyme A (CoA) and the reported structure is that of the CoA-bound form. We initially investigated the binding of CoA and acetyl CoA to PanZ using ITC; titration of CoA and AcCoA into PanZ showed binding in substoichiometric ratios but with micromolar affinity in both cases (Fig. 2, Table 1). We next investigated the interaction of PanZ with the ADC-T57V site-directed mutant by titration of PanZ into PanD, for which a sub-stoichiometric interaction was again observed (N = 0.616). The affinity of the interaction was however tight, with a fitted dissociation constant of 58 ± 11 nM. A reverse titration in which ADC-T57V was titrated into PanZ yielded a similar value for the dissociation constant (120 ± 30 nM) but an apparent super-stoichiometric number of binding sites (N = 1.33).


Formation of a heterooctameric complex between aspartate α-decarboxylase and its cognate activating factor, PanZ, is CoA-dependent.

Monteiro DC, Rugen MD, Shepherd D, Nozaki S, Niki H, Webb ME - Biochem. Biophys. Res. Commun. (2012)

Electrospray MS analysis of PanD-T57V and its complex with PanZ-CoA. (A) ESMS spectrum of PanD-T57V tetramer. (B) ESMS spectrum of PanD-T57V in the presence of PanZ-CoA reveals a mixture of species corresponding to a tetramer of PanD-T57V bound to either 3 or 4 equivalents of PanZ-CoA. (C) Simulation of peak positions for PanD–PanZ complexes matches observed peak positions.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3473359&req=5

f0010: Electrospray MS analysis of PanD-T57V and its complex with PanZ-CoA. (A) ESMS spectrum of PanD-T57V tetramer. (B) ESMS spectrum of PanD-T57V in the presence of PanZ-CoA reveals a mixture of species corresponding to a tetramer of PanD-T57V bound to either 3 or 4 equivalents of PanZ-CoA. (C) Simulation of peak positions for PanD–PanZ complexes matches observed peak positions.
Mentions: Cort et al. (PDB: 2k5t) noted that after overexpression in E. coli, PanZ is purified as a mixture of free protein and ligand-bound to coenzyme A (CoA) and the reported structure is that of the CoA-bound form. We initially investigated the binding of CoA and acetyl CoA to PanZ using ITC; titration of CoA and AcCoA into PanZ showed binding in substoichiometric ratios but with micromolar affinity in both cases (Fig. 2, Table 1). We next investigated the interaction of PanZ with the ADC-T57V site-directed mutant by titration of PanZ into PanD, for which a sub-stoichiometric interaction was again observed (N = 0.616). The affinity of the interaction was however tight, with a fitted dissociation constant of 58 ± 11 nM. A reverse titration in which ADC-T57V was titrated into PanZ yielded a similar value for the dissociation constant (120 ± 30 nM) but an apparent super-stoichiometric number of binding sites (N = 1.33).

Bottom Line: The existence of a fifth essential protein for pantothenate biosynthesis in some enteric bacteria has recently been reported by Stuecker et al. [10] and Nozaki et al. (in press) [9].This protein, PanZ, catalyses the activation of the PanD zymogen to form ADC and is essential for prototrophic growth.These approaches reveal that the two proteins interact with nanomolar affinity in a CoA-dependent fashion to form a heterooctameric complex.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Leeds, Leeds LS2 9JT, UK.

Show MeSH
Related in: MedlinePlus