Structural Basis of the γ-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

<div><p>The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of l-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-d-glucitol and d-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of l-gulonate in an extended (or all-trans) conformation; l-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.</p> </div

Active site structures of mouse and human SMP30/GNL.

<p>(<b>A</b>) Mouse SMP30/GNL in the substrate-free form, (<b>B</b>) the mouse SMP30/GNL–1,5-AG complex, (<b>C</b>) the human SMP30/GNL–1,5-AG complex, (<b>D</b>) the mouse SMP30/GNL–d-glucose complex, and (<b>E</b>) 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.</p

Top view of the substrate-binding cavity.

<p>Surface representation of (<b>A</b>) mouse SMP30/GNL, (<b>B</b>) DFPase, (<b>C</b>) Drp35, and (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053706#pone.0053706-Krissinel1" target="_blank">[27]</a> in the CCP4 program suite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053706#pone.0053706-Collaborative1" target="_blank">[20]</a>, and all molecules are viewed from the same direction.</p

Overall structure of mouse SMP30/GNL.

<p>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.</p

Structural comparison of the lid loops of mouse and human SMP30/GNL.

<p>(<b>A</b>) 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>B, C</b>) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse (<b>B</b>) and human (<b>C</b>) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. (<b>D</b>) 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.</p

Crystallographic data summary of human SMP30/GNL.

*<p>Values for the highest resolution shell are shown in parenthesis.</p

Crystallographic data summary of mouse SMP30/GNL.

*<p>Values for the highest resolution shell are shown in parenthesis.</p

Catalytic reaction of mouse SMP30/GNL.

<p>(<b>A</b>) 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>B–D</b>) Substrate and product analogues used in this study: (<b>B</b>) xylitol, (<b>C</b>) 1,5-anhydro-d-glucitol (1,5-AG), and (<b>D</b>) d-glucose. Corresponding atoms in these molecules are marked in light orange and light yellow.</p