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Results: 6

1.
FIG 6

FIG 6 . From: PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD).

Yeast two-hybrid analysis. Growth of yeast two-hybrid reporter strain AH109 carrying bait and prey plasmids encoding proteins indicated to the right of the image. Overnight cultures of each strain were normalized to an OD600 of 0.1 and then serially diluted and spotted on SD medium lacking histidine.

Tara N. Stuecker, et al. mBio. 2012 Jul-Aug;3(4):e00158-12.
2.
FIG 1

FIG 1 . From: PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD).

CoA-binding site of PanM. (A) Alignment of CoA-binding motif (motif A; highlighted in red) from Tetrahymena GCN5, human PCAF, and S. enterica PanM. The catalytic glutamate of tGCN5 and hPCAF is indicated by a black dot. PanM residue Gly76 is identified by an arrow. (B) Residues of PanM that interact with CoA. Side chain interactions are boxed; all other interactions involve main chain atoms.

Tara N. Stuecker, et al. mBio. 2012 Jul-Aug;3(4):e00158-12.
3.
FIG 2

FIG 2 . From: PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD).

Acetyl-CoA stimulates PanM activity by an acetyltransferase-independent mechanism. (A) Specific activities of PanD after preincubation with PanM alone (bar 1; gray), PanM with CoA (bar 2; gray), PanM with acetyl-CoA (bar 3; red), or PanM with ethyl-CoA (bar 4; blue). (B) Acetylation assays containing [1-14C]acetyl-CoA and pro-PanD alone (reaction 1) or pro-PanD with PanM (reaction 2). Proteins were visualized using Coomassie brilliant blue dye, and acetylation was detected by phosphorimaging. Proteins present in acetylation reaction mixtures were resolved using a Bio-Rad TGX Any kD gel (top panels) to visualize the C-terminal α fragment of mature PanD or a Bio-Rad 4 to 20% TGX gel to visualize the N-terminal β-fragment (bottom panels).

Tara N. Stuecker, et al. mBio. 2012 Jul-Aug;3(4):e00158-12.
4.
FIG 3

FIG 3 . From: PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD).

Alanine scan of PanM. Potential catalytic residues are E25 (blue), Y26 (green), S27 (purple), D64 (yellow), S65 (red), E103 (brown), and D104 (teal). (A) Stereo view of PanM bound to CoA (PDB 2K5T). Distances (Å) between each residue and the sulfur atom of CoA are indicated. CoA is colored by atom: C (pink), S (yellow), H (white), N (blue), and O (red). (B) Complementation of a ΔpanM S. enterica strain (JE12555) with panM genes encoding alanine substitutions at each potential catalytic residue. Final optical densities are shown for strains grown for 15 h on minimal glycerol medium lacking β-alanine. 5× variant, PanME25A, Y26A, S27A, D64A, S65A; 7× variant, PanME25A, Y26A, S27A, D64A, S65A, E103A, D104A.

Tara N. Stuecker, et al. mBio. 2012 Jul-Aug;3(4):e00158-12.
5.
FIG 4

FIG 4 . From: PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD).

Complementation of ΔpanD (JE13988; gray) and ΔpanM (JE13226; red) S. enterica strains with panD genes encoding AcK-pro-PanD variants. (A) Final optical densities of strains grown for 15 h on minimal glycerol medium lacking β-alanine. wt, wild type. (B) Western blot analysis of ΔpanM strains grown in minimal glycerol medium containing β-alanine using rabbit polyclonal antibodies elicited against pro-PanD. All proteins in cell lysates were resolved under native PAGE conditions, with PanD migrating as a tetramer. Lane 1 contained a Precision Plus All Blue protein ladder (Bio-Rad).

Tara N. Stuecker, et al. mBio. 2012 Jul-Aug;3(4):e00158-12.
6.
FIG 5

FIG 5 . From: PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD).

The CoA-binding-deficient PanMG76L variant. (A) Complementation of ΔpanM S. enterica (JE12555) in minimal glycerol medium with panM encoding either wild-type PanM (squares; black) or PanMG76L (triangles; red). (B) Western blot analysis of strains from panel A grown in minimal glycerol medium containing β-alanine. PanM was detected using monoclonal antibodies raised against the maltose binding protein (MBP) tag fused to the N terminus of PanM. MBP-PanM bands were quantified using densitometry analysis and normalized to the wild-type sample. The SEM for two separate experiments was 5.9%. Lane 1 contained the Precision Plus All Blue protein ladder (Bio-Rad). (C) Specific activities of PanD after preincubation with either PanMwt or PanMG76L. Reactions were carried out in the absence of any CoA derivatives (gray) or with 100 µM acetyl-CoA (ac-CoA) (red).

Tara N. Stuecker, et al. mBio. 2012 Jul-Aug;3(4):e00158-12.

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