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

Shingo Aizawa; Miki Senda; Ayaka Harada; Naoki Maruyama; Tetsuo Ishida; Toshiro Aigaki; Akihito Ishigami; Toshiya Senda

doi:10.1371/journal.pone.0053706

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.

Authors

Shingo Aizawa¹, Miki Senda, Ayaka Harada, Naoki Maruyama, Tetsuo Ishida, Toshiro Aigaki, Akihito Ishigami, Toshiya Senda

Affiliation

¹ Cellular Genetics, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo, Japan.

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.

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

Substances

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

Grants and funding

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 (http://www.jsps.go.jp/english/e-grants/index.html), and Grant-in-Aid for Scientific Research on Innovative Areas (22121005 TS) from the Ministry of Education, Science, and Culture, Japan (http://www.mext.go.jp/english/). This work was also supported by the New Energy and Industrial Technology Development Organization (NEDO) (http://www.nedo.go.jp/english/index.html) (P08005).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.