Structural basis of the γ-lactone-ring formation in ascorbic acid biosynthesis by the senescence marker protein-30/gluconolactonase.
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.
Cellular Genetics, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo, Japan.
Shingo
S
Aizawa
Miki
M
Senda
Ayaka
A
Harada
Naoki
N
Maruyama
Tetsuo
T
Ishida
Toshiro
T
Aigaki
Akihito
A
Ishigami
Toshiya
T
Senda
D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D018360:Crystallography, X-Ray; D005947:Glucose / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D007783:Lactones / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D014993:Xylitol / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D047908:Intracellular Signaling Peptides and Proteins; D007783:Lactones; C513323:RGN protein, human; C513324:Rgn protein, mouse; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase; D005947:Glucose; D001205:Ascorbic Acid; D014993:Xylitol