Table 1.
Data collection and refinement statistics.
Fig 1.
Overall structure of GDHNAD∙Gly.
(A) Overall structure of two protomers in the GDHNAD∙Gly structure. Mol-A and Mol-B are colored orange and purple, respectively. NAD+ and glycerol are shown in stick, and Zn2+ is shown as a grey sphere. (B) Structural superposition of the two protomers, showing the ligands on the central cleft. Mol-A and Mol-B are colored as in (A), and the ligands of each protomer are presented in stick.
Fig 2.
Ligand binding of two protomers of GDHNAD∙Gly.
(A) Mol-A of the GDHNAD∙Gly structure with simulated annealing omit Fo-Fc maps for each ligand contoured at 3.0 σ. The N- and C- terminals are marked as brown spheres. NAD+ and glycerol are shown in stick, and Zn2+ is shown as a grey sphere. (B) Schematic representation of the interactions between the ligands and their surroundings in Mol-A within the GDHNAD∙Gly structure. Hydrogen bonds are shown as dashed lines, and the participating residues are circled in yellow. Residues participating in hydrophobic interactions are shown with curved lines. (C) Mol-B in the GDHNAD∙Gly structure with a simulated annealing omit Fo-Fc map for NAD+. The two terminals and the ligands are shown as in (A). (D) Schematic representation of the interactions between NAD+ and its surroundings in Mol-B within the GDHNAD∙Gly structure shown as in (B). (E) Specific activities of E. coli GDH under four conditions; in the presence of NAD+, enzyme activity was measured with or without glycerol at pH 4.2 or 10.0. Data are shown as the mean ± 95% confidence intervals (95CI) for triplicate experiments. (F) Conformational comparisons between NAD+ (GDHNAD∙Gly) and NADH (from a horse liver alcohol dehydrogenase structure; PDB: 8G41) or between glycerol (GDHNAD∙Gly) and DHA (from a DHA kinase structure; PDB: 3PNQ) shown with their simulated annealing omit Fo-Fc maps contoured at 3.0 σ or their 2Fo—Fc maps contoured at 1.0 σ (green meshes).
Fig 3.
Oligomeric states of GDH crystal structures from various species.
(A) Phylogenetic analysis of the E. coli GDH structure and reported GDH structures from eight other species. The aligned sequences were plotted using Interactive Tree of Life (iTOL). Sequence identities were compared with E. coli GDH, and oligomeric states of each GDH crystal structure are presented (PDB: 5ZXL, 6CSJ, 5XN8, 4MCA, 1KQ3, 3UHJ, 1JQA, and 1TA9, in order). (B) GDH octamers from the eight reported crystal structures as in (A). The unit protomer is colored green. The 4-fold rotational axes in the structures are presented as black diamonds.
Fig 4.
Octameric interfaces of GDHNAD∙Gly.
(A) Octameric structure of GDHNAD∙Gly. Mol-A and Mol-B are colored orange and purple, respectively. Three octameric interfaces per protomer are shown as red surfaces in Mol-A. (B and C) Structural superposition of four E. coli GDH structures, including the GDHNAD∙Gly structure (B), and three B. stearothermophilus GDH structures (C). Mol-A of the GDHNAD∙Gly structure in (B) is colored orange. Regions showing major structural differences are indicated by dashed circles. The 4-fold rotational axes in the structures are presented as black diamonds.
Fig 5.
Details of the octameric interfaces in GDHNAD∙Gly.
(A) Overall structure of Mol-A within the GDHNAD∙Gly structure. The three octameric interfaces are shown as red surfaces. (B) Detailed views of the three octameric interfaces within the GDHNAD∙Gly structure. Mol-A, Mol-B, and the neighboring protomers are colored orange, purple, and white, respectively. The side or main chains of the residues participating in the interactions are shown in stick with dashed lines indicating hydrogen bonds.
Fig 6.
Structural comparison between E. coli and C. acetobutylicum GDHs.
(A) Structural superposition of Mol-As of the GDH structures from E. coli (GDHNAD∙Gly) and C. acetobutylicum (PDB: 1TA9) colored orange and green, respectively. The three octameric interfaces of GDH are marked as black circles. (B-D) Stereo views (wall-eye) of the three octameric interfaces of the superimposed structure in (A). For E. coli GDH, neighboring protomers are presented together with transparency, and the residues participating in the interface interactions are shown in stick as in Fig 5B. The residues corresponding to the interface residues in C. acetobutylicum GDH and E. coli GDH are shown together.
Fig 7.
Comparison of the structural dynamics of the GDH monomer and octamer.
(A-B) RMSFs per residue calculated from the MD simulations with the GDH monomer (A) and octamer (B) models in the apo (black), NAD+-bound (blue), and NAD+∙glycerol-bound (red) states. N-domain regions are marked with red boxes. (C-E) Averaged RMSFs for the model overall, the N-domain, and the C-domain from (A) and (B) in the apo (C), NAD+-bound (D), and NAD+∙glycerol-bound (E) states. Data are shown as the mean ± 95CI for triplicate experiments.
Fig 8.
Comparison of distances between ligands in the GDH monomer and octamer.
(A and B) Superposition of the 20 representative structures clustered from the results of the MD simulations with the GDH monomer (A) and octamer (B) models in the NAD+∙glycerol-bound state. NAD+ and glycerol are shown in stick, and Zn2+ is shown as a grey sphere. Detailed views of the active sites of each structure are presented with the distance ranges between the hydrogen atom of C2 of glycerol and the C4N atom of NAD+. (C) Distances between the hydrogen atom of C2 of glycerol and the C4N atom of NAD+ from (A) and (B) are plotted with curves calculated using Gaussian distribution fitting.