Fig 1.
Structures of enzyme substrates and products.
Numbers shown at myo-inositol and L-glucose denote the positions of hydroxyl group.
Table 1.
Data collection and refinement statistics.
Table 2.
Primers used for mutation.
Fig 2.
Overall structure of Pl-scyllo-IDH.
(A) The overall structure of Pl-scyllo-IDH is shown as ribbon model. The structure adopts a homotetrameric conformation. In the structure, L-glucono-1,5-lactone and NADH as shown as sphere and stick models, respectively. A loop region colored in red in the yellow colored subunit protrudes to the active site in the next grey colored subunit, circled with a blue dotted line, described in detail in the text. (B) Subunit structure of Pl-scyllo-IDH shown as a ribbon model. Colors are assigned based on the secondary structure. NADH-binding and substrate-binding domains are colored yellow with red, and magenta with cyan, respectively. L-glucono-1,5-lactone and NADH are shown as stick models colored in blue. The loop region extending to the next subunit is marked with a blue dotted circle, which also corresponds to the circle in panel A.
Fig 3.
Binding modes of Pl-scyllo-IDH substrates.
The binding modes of L-glucono-1,5-lactone (A), myo-inositol (B) and scyllo-inosose (C) are shown as stick models. The substrates and amino acids are colored yellow and green, respectively. His318, colored in cyan, belongs to the adjacent subunit. NAD+/NADH are also shown in a wireframe model colored in green. The dotted lines indicate the distances below 3.2 Å, with the exception of the 3.4 Å distance between Y163 and O2 in myo-inositol (B). Electron density of the Fo-Fc omit map contoured at 2σ, calculated excluding corresponding molecules, are also shown at the right side in each panel. In (A) and (B), right panels, the arrows indicate the viewing directions of the models, and the labels corresponds to the top in each panel. The red colored arrow b in (A) indicates a carbonyl group. In (C), the arrows show the position of a carbonyl group in scyllo-inosose.
Fig 4.
Sequence alignment of structurally identified GFO/IDH/MocA family proteins.
The amino acid sequence alignment was performed using ClustalW. Proteins are represented by PDB codes as follows: 4FB5; a probable oxidoreductase from Rhizobium etli, 1OFG; a glucose-fructose oxidoreductase from Zymomonas moblis, 3NT2; a myo-IDH from B. subtilis and 4N54; a scyllo-inositol DH from Lactobacillus casei. The three red arrow heads show the proposed catalytic residues. The black arrow heads show residues involved in substrate binding in Pl-scyllo-IDH. The blue arrow head indicates H318 in Pl-scyllo-IDH, which is involved in the substrate binding in the adjacent subunit. The green line indicates the flexible loop region covering the active site in Pl-scyllo-IDH. Arg178 located in the loop region is colored in red. The orange line shows the loop region involved in the subunit interaction in Pl-scyllo-IDH.
Fig 5.
Superposition of flexible loop regions in each complex.
The Pl-scyllo-IDH structures complexed with L-glucono-1,5-lactone, myo-inositol, scyllo-inosose, and acetate are shown superimposed, and the flexible loop regions are shown. Arg178 in the loop is also shown as a stick model. The regions colored in cyan and magenta indicate myo-inositol and scyllo-inosose, and L-glucono-1,5-lactone and acetate, respectively.
Table 3.
Kinetic parameters of Pl-scyllo-IDH with L-2-epi-inosose and substrates.
Table 4.
Kinetic parameters of Pl-scyllo-IDH mutants with sycllo-inositol and L-glucose.
Fig 6.
(A) L-glucono-1,5-lactone (yellow) and NADH (green) bound in Pl-scyllo-IDH are shown as stick models. β-L-glucose, colored in cyan, is superposed on the lactone molecule, and the H1 hydrogen in the axial conformation is colored white. The distance between NC4 and C1 is 3.4 Å, and the distance between H1 and NC4 is 2.0 Å shown as a red dotted line. From this structure, Pl-scyllo-IDH is concluded to be an A-type enzyme. (B) Structures of each of chain B in Pl-scyllo-IDH complexed with L-glucono-1,5-lactone colored in green and pale green, and myo-IDH from B. subtilis complexed with scyllo-inosose (PDB code: 3NT5) colored in orange and pale orange were superposed using the program Chimera [25]. The superposed active sites are shown with substrates, NADH, and the catalytic triad, K-D-H in stick models. The difference is observed in the orientations of the nicotinamide rings between Pl-scyllo-IDH and myo-IDH, which represent the A-type and B-type enzymes, respectively.
Fig 7.
Phylogenetic tree of GOFR/IDH/MocA family members present in PDB.
The amino acid sequences of GFO/IDH/MocA family enzymes in PDB were aligned and then subjected to a phylogenetic analysis using the program MEGA7. The number after PDB code indicates the oligomeric state of enzyme. A red line shows structures harboring the swap loop region as observed in Pl-scyllo-IDH.