Volume 9, Issue 5, Pages 439-450 (May 2001) The Crystal Structure of E. coli Pantothenate Synthetase Confirms It as a Member of the Cytidylyltransferase Superfamily  Frank von Delft, Ann Lewendon, Venugopal Dhanaraj, Tom L. Blundell, Chris Abell, Alison G. Smith  Structure  Volume 9, Issue 5, Pages 439-450 (May 2001) DOI: 10.1016/S0969-2126(01)00604-9

Figure 1 The Pantothenate Pathway in E. coli Structure 2001 9, 439-450DOI: (10.1016/S0969-2126(01)00604-9)

Figure 2 General Structure (a) Representative experimental electron density (solvent-flattened MAD phases) and final, refined, atomic model of the phosphate binding KMSKS motif residues in subunit A. (b) Stereo Cα-trace of one PS subunit, colored by position in the sequence, with every tenth residue numbered. (c) Stereo cartoon representation of the PS dimer, with subunits colored differently, and secondary structure numbered as in (d). The view is approximately along the two-fold axis (shown as a dot) relating the N-terminal domains. The figure was prepared with MOLSCRIPT [53]. (d) Schematic diagram of the secondary structural elements of one PS subunit; β strands are shown in blue, α helices are shown in yellow, and 310 helices are shown in orange. For the Rossmann domain, βα pairs have the same number, and structural elements of the dimerization insertion are denoted by D. P176 denotes the approximate domain boundary Structure 2001 9, 439-450DOI: (10.1016/S0969-2126(01)00604-9)

Figure 3 Structural Alignment and Superposition (a and b) Superposition of the PS domain N (red) with the matching secondary structure elements from (a), CGT (blue) and PPAT (gold), and from (b), QtRS (light blue) and EtRS (green). Also shown is ATP found in QtRS. The figure was prepared with MOLSCRIPT [53]. (c) Structure-guided alignment of the sequences of the homologous Rossmann domains, in JOY format [52]; this is a more complete and accurate alignment than the purely sequence-based alignment of Bork et al. [9]. The HIGH and KSMKS motifs are indicated by large boxes, and residues in QtRS, CGT, and PPAT that interact with ATP (or CTP) are indicated by small boxes. PS residues proposed to contact ATP are in shaded boxes and marked for complete (solid circle) or partial (asterisk) conservation among orthologous PS sequences. The three residues shaded in blue are in different one-dimensional, but equivalent three-dimensional positions. (See http://www.cryst.bioc.cam.ac.uk/~joy for key to symbols.) Structure 2001 9, 439-450DOI: (10.1016/S0969-2126(01)00604-9)

Figure 4 The Reactions of the Superfamily Comparisons of the reactions catalyzed by PS [2,3], QtRS [23], CGT [28], and PPAT [27]. The step shown is fundamentally the same for all reactions: attack of some nucleophilic, acidic oxygen atom on the α-P atom of a nucleotide triphosphate to release pyrophosphate. For PS and QtRS (and the other tRS), this is the first of two reaction steps; for PPAT and CGT, there is only one step Structure 2001 9, 439-450DOI: (10.1016/S0969-2126(01)00604-9)

Figure 5 Domain Movement (a) The orientation of the PS C-terminal domain (yellow) relative to the N-terminal domain (bright red) in the crystal structure. (b) The orientation of the PS C-terminal domain (yellow) relative to the N-terminal domain (bright red) after the modeled rotation (arrow in [a]) that aligns helix 3107 (dark red ribbon) with helix αI of QtRS (dark green ribbon). For both PS and QtRS, KMSKS Cα atoms are shown as spheres (gray and light green, respectively), and residues of strand β5 up to the helices are shown as Cα-trace (dark red and dark green, respectively). PS residues that were rotated are blue in (b). ATP (orange) is shown in (a), in the way it is bound to QtRS that has been aligned to PS, and in (b), in its position after minimization; it binds against the HIGH residues (pink spheres on helix α1 in [a]). The position of the positive charge that binds Pα in QtRS (K270, dark green in [a]) is retained in this model of PS (S188 and Mg2+, gray and light blue, respectively, in [b]), and R189 (shown but not labeled) may also bind Pα. The figure was prepared with MOLSCRIPT [53] Structure 2001 9, 439-450DOI: (10.1016/S0969-2126(01)00604-9)

Figure 6 Substrate Binding Model (a) Proposed ATP binding interactions. (b) Proposed binding of ATP and pantoate in the active site. The molecular surface (green) was calculated after energy minimization of the modeled PS-ATP complex, and the pocket appears perfect for the bulky pantoate. The apparent “clash” with the surface is caused by the H bonding of the pantoate carboxylate to both Q61 and Q155, since the surface was calculated with hydrogen atoms added. The putative line of attack by pantoate is shown (red dotted line). The figure was prepared with SYBYL [31]. (c) Schematic diagram of pantoate-PS interactions. Note the cluster of conserved, hydrophobic residues to accommodate the two methyl groups. Both hydroxy groups can be accommodated, and the carboxylate group is at a suitable distance for attack Structure 2001 9, 439-450DOI: (10.1016/S0969-2126(01)00604-9)