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PMID: 19369590 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Galactonolactone dehydrogenase requires a redox-sensitive thiol for optimal production of vitamin C.

Plant physiology ·Vol. 150 ·No. 2 ·2009-06-00 ·Pages 596-605

Leferink NG, van Duijn E, Barendregt A, Heck AJ, van Berkel WJ

Abstract

The mitochondrial flavoenzyme l-galactono-gamma-lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340, located in the cap domain. Electrospray ionization mass spectrometry revealed that the partial reversible oxidative modification of Cys-340 involves the sequential formation of sulfenic, sulfinic, and sulfonic acid states. S-Glutathionylation of the sulfenic acid switches off GALDH activity and protects the enzyme against oxidative damage in vitro. C340A and C340S GALDH variants are insensitive toward thiol oxidation, but exhibit a poor affinity for l-galactono-1,4-lactone. Cys-340 is buried beneath the protein surface and its estimated pK(a) of 6.5 suggests the involvement of the thiolate anion in substrate recognition. The indispensability of a redox-sensitive thiol provides a rationale why GALDH was designed as a dehydrogenase and not, like related aldonolactone oxidoreductases, as an oxidase.

MeSH Terms
Amino Acid Sequence Arabidopsis/enzymology Ascorbic Acid/biosynthesis Catalytic Domain Cysteine/metabolism Electron Spin Resonance Spectroscopy Enzyme Activation Glutathione/metabolism Kinetics Molecular Sequence Data Oxidation-Reduction Oxidative Stress Oxidoreductases Acting on CH-CH Group Donors/chemistry,metabolism Protein Folding Sequence Homology, Amino Acid Spectrometry, Mass, Electrospray Ionization Spin Labels Sulfhydryl Compounds/metabolism Time Factors
Chemicals
Spin Labels Sulfhydryl Compounds Oxidoreductases Acting on CH-CH Group Donors galactonolactone dehydrogenase Glutathione Cysteine Ascorbic Acid
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Leferink Nicole G H
Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands.
van Duijn Esther
Barendregt Arjan
Heck Albert J R
van Berkel Willem J H
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Article Info
Journal
Plant physiology
Abbr.
Plant Physiol
ISSN
0032-0889
DOI
10.1104/pp.109.136929
Published
2009-06-00
Epub
2009-00-15
Pages
596-605
Language
English
Region
United States
NLM ID
0401224
PMCID
PMC2689977
Subset
IM
External Links
PubMed source DOI: 10.1104/pp.109.136929 PMC Full Text View journal details
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