The missing step of the L-galactose pathway of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increases leaf ascorbate content

Abstract

The gene for one postulated enzyme that converts GDP-L-galactose to L-galactose-1-phosphate is unknown in the L-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes D-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-L-galactose to L-galactose-1-P. The expressed protein is best described as a GDP-L-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-l-galactose to a hexose-1-P, most likely D-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-L-galactose-D-mannose-1-phosphate guanyltransferase activity.

Fig. 1.

An alignment of the A. thaliana sequence VTC2 with the kiwifruit sequence 319998 and a second A. thaliana sequence At5 g55120. Also shown are the Arabidopsis enzyme At5 g18200 [coding for a putative UDP-d-glucose-hexose-1-phosphate uridylyltransferase (EC 2.7.7.12)] and the unnamed mouse protein Mm_74150758 (the GenBank accession no.). Identical aligned residues in all five sequences are shown in dark gray, similar residues in light gray. The sequences were aligned by using Clustal X (41) with some manual adjustment. The HIT triad sequence is identified at approximately amino acid residue 250.

Fig. 2.

Response of the GDP-mannose-1-P guanyl transferase to GDP-l-galactose. GDP-l-galactose was made from GDP-d-mannose by using the epimerase as described was in Methods, and the concentration that was GDP-l-galactose in the mixture was determined by HPLC. Assays were conducted by using the continuous coupled assay by using 0.029 μg of enzyme per assay. Mannose-1-P concentration was 0.93 and 1.87 mM MgCl₂. Other conditions were as described in the text. Squares represent the reaction minus the background run without mannose-1-P. Triangles represent the background values by using HisTrap purified E. coli extract (0.006 μg) expressing an empty PET30a vector.

Fig. 3.

Response of the enzyme to potential guanyl acceptors. Assays were carried out by using the continuous coupled assay with varying concentrations of inorganic phosphate (square), inorganic pyrophosphate (circle), or d-mannose-1-P (triangle) as the guanyl acceptor. The V_max values were 0.12 ± 0.03, 0.032 ± 0.002, and 0.17 ± 0.009 nmol sec⁻¹·μg⁻¹ protein for the substrates phosphate, pyrophosphate, and d-mannose-1-P, respectively. K_m values were 4.4 ± 2, 0.16 ± 0.05, and 0.11 ± 0.03 mM, respectively. Assays were carried out three times with similar results.

Fig. 4.

Effect of transiently expressed 319998 on ascorbate content and enzyme activity in tobacco leaves. See Methods for details. White bars represent ascorbate concentration (expressed on a fresh-weight basis) in the leaf, and black bars represent the GDP-l-galactose D-mannose-1-phosphate guanyltransferase activity (expressed on a gram of protein basis). L1, L2, and L3 represent the three youngest leaves that were injected. Error bars are the standard errors of the mean (n = 3–6).

Fig. 5.

Reactions converting d-mannose-1-phosphate to l-galactose-1-phosphate. Reactions in boxes represent summations of the reactions above them.