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l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxi-doreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thalianaGALDH (AtGALDH). | ỊFEBS Journal L-Galactono-Ỵ-lactone dehydrogenase from Arabidopsis thaliana a flavoprotein involved in vitamin C biosynthesis Nicole G. H. Leferink Willy A. M. van den Berg and Willem J. H. van Berkel Laboratory of Biochemistry Wageningen University the Netherlands Keywords Arabidopsis thaliana flavoprotein L-galactono-1 4-lactone dehydrogenase site-directed mutagenesis vitamin C biosynthesis Correspondence W. J. H. van Berkel Laboratory of Biochemistry Wageningen University Dreijenlaan 3 6703 HA Wageningen the Netherlands Fax 31 317 484801 Tel 31 317 484468 E-mail willem.vanberkel@wur.nl Website http www.bic.wur.nl Received 10 September 2007 revised 14 November 2007 accepted 12 December 2007 doi 10.1111 j.1742-4658.2007.06233.x L-Galactono-1 4-lactone dehydrogenase GALDH ferricytochrome c oxi-doreductase EC 1.3.2.3 is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C L-ascorbic acid in plants. In the present study we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH AtGALDH . AtGALDH oxidizes in addition to L-galactono-1 4-lactone Km 0.17 mM kcat 134 s 1 L-gulono-1 4-lactone Km 13.1 mM kcat 4.0 s 1 using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen kox 6 X 102 M_1-s_1 . Unlike most flavoprotein dehydrogenases AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding but contains a leucine instead Leu56 . Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for .
