Figure 1.
Catalytic reaction of mouse SMP30/GNL.
(A) The γ-lactone-forming reaction catalyzed by mouse SMP30/GNL. The product of the catalytic reaction of SMP30/GNL is l-gulono-γ-lactone, which is in turn converted to ascorbic acid by gluconolactone oxidase. (B–D) Substrate and product analogues used in this study: (B) xylitol, (C) 1,5-anhydro-d-glucitol (1,5-AG), and (D) d-glucose. Corresponding atoms in these molecules are marked in light orange and light yellow.
Table 1.
Crystallographic data summary of mouse SMP30/GNL.
Table 2.
Crystallographic data summary of human SMP30/GNL.
Figure 2.
Overall structure of mouse SMP30/GNL.
The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M2+) located at the center of the structure is shown as an orange sphere.
Figure 3.
Structural comparison of the lid loops of mouse and human SMP30/GNL.
(A) Lid loops of mouse and human SMP30/GNL in the substrate free form are shown in purple and blue, respectively. The divalent metal ion (labeled as M2+) is shown in orange. (B, C) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse (B) and human (C) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. (D) Surface representation of mouse SMP30/GNL around the lid loop. The entrance for the substrate-binding cavity is indicated by an arrow. Residues in the lid loop are shown in purple.
Figure 4.
Active site structures of mouse and human SMP30/GNL.
(A) Mouse SMP30/GNL in the substrate-free form, (B) the mouse SMP30/GNL–1,5-AG complex, (C) the human SMP30/GNL–1,5-AG complex, (D) the mouse SMP30/GNL–d-glucose complex, and (E) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.
Figure 5.
Top view of the substrate-binding cavity.
Surface representation of (A) mouse SMP30/GNL, (B) DFPase, (C) Drp35, and (D) PON. Residues in the lid loop of mouse SMP30/GNL and the divalent metal ions (labeled as M2+) are shown in purple and orange, respectively. Structures of DFPase, Drp35, and PON are superposed onto mouse SMP30/GNL by the SSM fitting using the program Superpose [27] in the CCP4 program suite [20], and all molecules are viewed from the same direction.
Figure 6.
Proposed catalytic reaction mechanism of mouse SMP30/GNL.
The divalent metal ion in the active site is labeled as M2+.