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   other side.
   [105]Figure 3
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   Figure 3

   Stereoview of AMPPNP binding. a, the F [o] −F [c] simulated annealing
   omit map of the ATP binding site of one subunit showing the bound
   AMPPNP. The map was calculated from 50.0 to 2.6 Ã… resolution and
   contoured at 3.0 Ï‚. Figs. [109]3 A and [110]4 A were prepared using
   XtalView ([111]18). b, hydrogen bonds are indicated by solid lines.
   Carbons are shown in yellow for AMPPNP andgray for bPanK. Nitrogens are
   shown in blue, oxygens are in red, phosphorus is in green, and Mg^2+ is
   in cyan. Asn^43 involved in ATP recognition by stacking interaction
   with the adenine base of AMPPNP is not labeled for clarity.

 §5§ CoA-binding Site §5§

   The well defined electron density for CoA is seen in both dimers
   occupying the asymmetric unit of the CoA-bound bPanK (Fig. [112]4 a).
   CoA is bound in the enzyme in a bent conformation in a deep pocket
   lined by residues from helix H, the P loop, the connecting loop of
   strands 5 and 6 and the connecting loop of helices H and I (Fig. [113]4
   b). Because CoA is a competitive inhibitor with respect to ATP and
   these two ligands share the ADP moiety, it was suggested that both
   ligands bind to the same site ([114]4, [115]11). Unexpectedly, the
   adenine base of CoA is inserted between the side chains of His^177 and
   Phe^247, whereas the adenine-interacting residues in the AMPPNP-bound
   enzyme are Asn^43 and His^307 (Fig.[116]4 b). The α-phosphate of CoA is
   salt bridged to the guanidinium group of Arg^243 and the amino group of
   Lys^101 that also interacts with the β-phosphate of CoA (Fig. [117]4
   b). The 3′-phosphate of CoA is hydrogen-bonded to the amide nitrogen of
   Ile^42 and the hydroxyl group of Ser^102 and salt bridged to the
   guanidinium group of Arg^106 (Fig. [118]4 b). These interactions of the
   3′-phosphate group may be essential for the inhibitory effect of CoA
   because dephospho-CoA is a significantly less potent inhibitor of bPanK
   activity ([119]4). By comparison, the same 3′-phosphate group in
   several CoA-binding proteins shows no interaction with the protein and
   is exposed to solvent (for review see Ref. [120]23). The exceptions are
   acyl-CoA binding protein ([121]24) and the surfactin synthetase
   activating enzyme Sfp ([122]25), in which the 3′-phosphate interacts
   with the protein residues such as His and Lys.
   [123]Figure 4
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   Figure 4

   Stereoview of coenzyme A binding. a, the F [o] −F [c] simulated
   annealing omit map of the CoA binding site of one subunit showing the
   bound CoA. The map was calculated from 50.0 to 2.5 Ã… resolution and
   contoured at 3.0 Ï‚.b, hydrogen bonds are indicated by solid lines. The
   thiol group of coenzyme A is shown in light green, and other atom
   colors are as described in the legend to Fig.[127]3 b. His^177 involved
   in CoA recognition by stacking interaction with the adenine base and
   Asn^282forming a hydrogen-bond with the β-mercaptoethylamine moiety are
   not labeled for clarity.

   Hydrophobic atoms of the pantothenate moiety of CoA form van der Waals'
   contacts with residues Leu^130, Tyr^175, and Ile^281 (Fig. [128]4 b).
   The carbonyl oxygen of the pantothenate moiety near the
   β-mercaptoethylamine moiety is hydrogen bonded to the hydroxyl group of
   Tyr^240 and to the amide group of Asn^282 (Fig. [129]4 b). The amide
   nitrogen of the β-mercaptoethylamine moiety is hydrogen-bonded to the
   hydroxyl group of Tyr^180 (Fig. [130]4 b). Strikingly, the thiol group
   of the β-mercaptoethylamine moiety is tightly sealed from water
   molecules by interacting with four aromatic residues, Phe^244, Phe^252,
   Phe^259, and Tyr^262 and also by intra-molecular hydrogen bonding to
   the amino group of the adenine base (Fig. [131]4 b). The thiol group
   approaches the face of Phe^259 at the distance of 3.8 Ã… between the
   sulfur atom and the ring centroid. The thiol group also approaches the
   edges of Phe^244, Phe^252, and Tyr^262, and the distances between the
   sulfur atom and the ring carbons of Phe^244, Phe^252, and Tyr^262 are
   3.8, 3.5, and 4.1 Ã…, respectively. These sulfur-aromatic interactions
   are weakly polar interactions ([132]26-30) that are stronger than van
   der Waals' interactions between nonpolar atoms ([133]31). These types
   of sulfur-aromatic interactions are involved in protein stability and
   function ([134]31, [135]32). The structure of another regulatory enzyme
   in the CoA biosynthetic pathway, phosphopantetheine
   adenylyltransferase, exhibits a dinucleotide-binding fold ([136]33).
   The C-terminal loops of five parallel β-strands bind the product
   dephospho-CoA. As in the CoA-bound bPanK, the thiol group of
   dephospho-CoA in phosphopantetheine adenylyltransferase is shielded
   from water molecules by interacting with the side chains of hydrophobic
   residues.

 §5§ The Specific Binding of Coenzyme A Compared with AMPPNP §5§

   Structural analysis of the AMPPNP-bound and the CoA-bound bPanKs
   provides the basis for understanding the CoA inhibition compared with
   the substrate ATP. Superimposed structures of the AMPPNP-bound and the
   CoA-bound enzymes (an average RMSD of 1.48 Å) shows that both the α-
   and β-phosphates of CoA and the β- and γ-phosphates of AMPPNP occupy
   the same space and interact with the residue Lys^101 (Fig. [137]5).
   This suggests that inhibition by CoA and its thioesters is achieved by
   occluding ATP binding to Lys^101. Mutation of this residue abolishes
   the binding of ATPγS, a nonhydrolyzable ATP analogue, as well as CoA to
   bPanK ([138]11), supporting the involvement of Lys^101 in binding of
   both ligands.
   [139]Figure 5
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   Figure 5

   Close-up stereoview of the overlapping site of two ligands, AMPPNP and
   CoA. The binding pockets of AMPPNP and CoA are shown in yellow and
   cyan, respectively. CoA (magenta) and AMPPNP (black) are shown inlines.
   a, residues of the AMPPNP-bound enzyme are shown in brown, and residues
   of the CoA-bound enzyme are not shown for clarity. Note that the
   carboxyl group of Glu^249of the AMPPNP-bound enzyme coincides with the