Abstract
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.
Highlights
Senescence marker protein-30 (SMP30) is a 34-kDa protein whose tissue levels in the liver, kidney, and lung decrease with aging [1,2]
While the crude extract supplemented with purified SMP30/ GNL showed the GNL activity, the GNL activity was not affected by changing the total amount of the crude extract in the reaction mixture (Table S2)
This result showed that SMP30/GNL is unlikely to function as an activator of the GNL activity in the contaminants
Summary
Senescence marker protein-30 (SMP30) is a 34-kDa protein whose tissue levels in the liver, kidney, and lung decrease with aging [1,2]. This protein is expressed in several organs but most prominently in the liver and kidney [1,3]. While biological analyses have suggested various homeostatic roles of regucalcin [7,8], these activities will not be discussed in the present manuscript. In this manuscript, we analyzed only the gluconolactonase activity of SMP30
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