(A) Ensemble of the eight lowest energy structures of the DHBPS monomer. Three loops (31–43, 84–94, and 176–185) and the N and C termini for which no long range NOE correlations were observed are marked in black. (B) Ribbon (MOLMOL; ref. 31) diagram of the DHBPS monomer.

Data from chemical shift mapping experiments investigating ligand binding and mutations with reduced activity mapped onto the three-dimensional structure depicted as a ribbon diagram (MOLMOL; ref. 31). Residues with strong (Δδ_TOTAL > 0.12 ppm) or moderate (moderate Δδ_TOTAL = 0.06–0.12 ppm) chemical shift perturbations after addition of ligands and complete or significant loss in activity after mutation are marked in dark and light colors, respectively. The conserved acidic loop joining strands β1 and β2 is indicated by green stripes. (A) The reaction catalyzed by DHBPS. (B) Chemical shift mapping of substrate binding to the enzyme in the absence of Mg²⁺ (nonturnover conditions). Strong and moderate shifts (see Fig. 3) are shown in blue and cyan, respectively. (C) Chemical shift mapping of product binding to the enzyme and catalysis in the presence of Mg²⁺ (turnover conditions). Strong and moderate shifts (see Fig. 3) are shown in red and yellow, respectively. (D) Results of site-directed mutagenesis experiments. Residues that, when substituted in the Methanococcus sequence, led to a loss of catalytic activity and those with a significant reduction in activity are shown in magenta and pink, respectively (see Table 2).

Two-dimensional 100-ms NOESY spectra of DHBPS. The regions show the aromatic protons (Lower) and long range correlations from aromatics to methyl protons (Upper). Spectra from a sample with 75% random fractional deuteration, ¹³C/¹⁵N labeling (A) and a sample with protonated Phe, Tyr, Thr, Ile, and Val in a fully protonated and ¹⁵N-labeled background (B) are shown.

Sequence alignments of four divergent members of the DHBPS family. Residues strictly conserved in 20 sequences are shown in bold (Ec, E. coli; Mj, M. jannaschii; Mt, Methanobacterium thermoautotrophicum; Af, Archaeoglobus fulgidus; A, Aeropyrum). The secondary structure of the E. coli enzyme is shown above the sequence. Results from NMR chemical shift mapping experiments investigating the binding of ligands is indicated by bars (red, blue, and green) above the sequence. The combined chemical shift perturbations (Δδ_TOTAL) of all ¹H and ¹⁵N resonances were weighted according to Δδ_TOTAL = Δδ [¹H] + 0.2 Δδ [¹⁵N] (20) and grouped into two classes (moderate, short bars Δδ_TOTAL = 0.06–0.12 ppm and strong, tall bars Δδ_TOTAL > 0.12 ppm). Red bars show the location and magnitude of chemical shift changes on the addition of the substrate, ribulose-5-phosphate, in the presence of Mg²⁺ ions. Blue bars indicate signals affected after the addition of substrate in the absence of Mg²⁺. Green bars indicate perturbations after the addition of Mg²⁺. The backbone nuclei of the residues that were not assigned in NMR spectra are marked with a dashed line in the secondary structure. Amino acids that, when substituted in the M. jannaschii sequence by site-directed mutagenesis, led to a loss of catalytic activity are highlighted in red, and those leading to significantly reduced activity are highlighted in cyan.

Space-filled representation (MOLMOL; ref. 31) of the dimer model built using the monomeric NMR structure. The model satisfies the intermolecular contacts reported by Liao et al. (6). The active site shown on the front of the dimer is formed by the regions showing large chemical shift changes on ligand binding: I, 32–42, green; II, 59–71, red; V, 150–155, magenta; VI, 170–176, cyan, from monomer A; III, 105–113, yellow; IV, 135–138, blue, from monomer B.