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J. Biol. Chem., Vol. 283, Issue 27, 18483-18492, July 4, 2008

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A Second GDP-L-galactose Phosphorylase in Arabidopsis en Route to Vitamin C

COVALENT INTERMEDIATE AND SUBSTRATE REQUIREMENTS FOR THE CONSERVED REACTION^*

Carole L. Linster¹, Lital N. Adler, Kristofor Webb, Kathryn C. Christensen, Charles Brenner, and Steven G. Clarke²

From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095 and the Departments of Genetics and Biochemistry, Dartmouth Medical School, Lebanon, New Hampshire 03756

The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.

Received for publication, April 3, 2008 , and in revised form, May 1, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM026020 (to S. G. C.), AG018000 (to S. G. C.), and CA75954 (to C. B.). This work was also supported by National Science Foundation Grant MCB-0448533 (to S. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a research fellowship (bourse de formation-recherche) from the Luxembourgish Government.

² To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, 607 Charles E. Young Dr. E., Los Angeles, CA 90095. Tel.: 310-825-8754; Fax: 310-825-1968; E-mail: clarke{at}mbi.ucla.edu.

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