Structural basis of the γ-lactone-ring formation in ascorbic acid biosynthesis by the senescence marker protein-30/gluconolactonase

PLoS One. 2013;8(1):e53706. doi: 10.1371/journal.pone.0053706. Epub 2013 Jan 22.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ascorbic Acid / biosynthesis*
  • Ascorbic Acid / chemistry*
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / metabolism*
  • Carboxylic Ester Hydrolases / chemistry
  • Carboxylic Ester Hydrolases / metabolism*
  • Cations, Divalent / metabolism
  • Crystallography, X-Ray
  • Glucose / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Lactones / chemistry*
  • Lactones / metabolism*
  • Mice
  • Models, Molecular
  • Protein Conformation
  • Xylitol / metabolism


  • Calcium-Binding Proteins
  • Cations, Divalent
  • Intracellular Signaling Peptides and Proteins
  • Lactones
  • RGN protein, human
  • Rgn protein, mouse
  • Carboxylic Ester Hydrolases
  • gluconolactonase
  • Glucose
  • Ascorbic Acid
  • Xylitol

Grant support

This work was supported by Grant-in-Aid for Scientific Research (B) (24380073 AI), Grant-in-Aid for Scientific Research (C) (23590441 NM) from Japan Society for the Promotion of Science (, and Grant-in-Aid for Scientific Research on Innovative Areas (22121005 TS) from the Ministry of Education, Science, and Culture, Japan ( This work was also supported by the New Energy and Industrial Technology Development Organization (NEDO) ( (P08005).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.