Galactonolactone dehydrogenase requires a redox-sensitive thiol for optimal production of vitamin C.
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.
Leferink
Nicole G H
NG
Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands.
van Duijn
Esther
E
Barendregt
Arjan
A
Heck
Albert J R
AJ
van Berkel
Willem J H
WJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001205:Ascorbic Acid / Q000096:biosynthesis*; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013997:Time Factors
D013113:Spin Labels; D013438:Sulfhydryl Compounds; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid