Partial purification and identification of GDP-mannose 3",5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway

Abstract

The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of l-galactosyl residues, catalyzed by a largely unknown GDP-d-mannose 3",5"-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-d-mannose 3",5"-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a blast search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-d-mannose 5"-epimerase. The plant GDP-d-mannose 3",5"-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.

Figure 1

Proposed de novo pathway for the synthesis of l-AA from d-Man in plants. Enzymes 1, hexokinase; 2, phosphomannomutase; 3, GDP-Man pyrophosphorylase; 4, GDP-Man 3",5"-epimerase; 5, l-Gal dehydrogenase; 6, l-galactono-1,4-lactone dehydrogenase.

Figure 2

Correlation between l-AA content and GDP-Man 3",5"-epimerase activity during the growth of A. thaliana cell suspension. Total l-AA content of cells and specific epimerase activity of the corresponding 55–70% ammonium sulfate fractions were determined as described in Materials and Methods.

Figure 3

Purification of GDP-Man 3",5"-epimerase from A. thaliana suspension cells. (A) DEAE-Sepharose FPLC of the ammonium sulfate fraction. The enzyme was eluted with a linear gradient of NaCl from 0 to 200 mM (see Materials and Methods). (B) Sephacryl S-200 gel filtration chromatography of epimerase fractions from DEAE-Sepharose. (C) Hydroxylapatite FPLC of epimerase fractions from Sephacryl gel filtration. The proteins were eluted with a linear gradient of 2–500 mM potassium phosphate (pH 7.2) (see Materials and Methods).

Figure 4

SDS/PAGE of the partially purified GDP-Man 3",5"-epimerase. Proteins were visualized by Coomassie blue staining. (Left) Molecular mass standards. (Right) NAD-eluted fraction from Blue-Sepharose. Proteins were identified by MS of in-gel tryptic digests. Band 1, betaine aldehyde dehydrogenase; band 2, glutathione synthase; bands 3 and 4, GDP-Man 3",5"-epimerase, with apparent molecular masses of 46 and 43 kDa, respectively.

Figure 5

Nano-ESI MS/MS spectrum of the m/z 785.9 [M + 2H]⁺² peptide ion derived from in-gel tryptic digestion of the 46- and 43-kDa bands of GDP-Man 3",5"-epimerase (see Fig. 4).

Figure 6

Database amino acid sequence of a hypothetical epimerase/dehydratase-like protein of A. thaliana (European Molecular Biology Laboratory accession no. AF272706) corresponding to the purified GDP-Man 3",5"-epimerase. The thick underlined region corresponds to the m/z 785.9 peptide sequence obtained by the nano-ESI MS/MS of the in-gel tryptic digest of the 46- and 43-kDa epimerase bands (see Fig. 5). Peptide ions corresponding to the underlined regions were observed by MALDI-TOF MS (see Table 2).

Figure 7

Multiple sequence alignment of GDP-Man 3",5"-epimerase from A. thaliana with related proteins detected by PSI-blast. The alignment was created with CLUSTALX (40), using default parameters, and displayed with the POSTSCRIPT VIEWER program. Colors follow CLUSTALW convention (41). EPIM, GDP-Man 3",5"-epimerase from A. thaliana; BlmG, NDP-d-Man 5"-epimerase of S. verticillus; SYNECHOCYSTIS, a hypothetical protein of Synechocystis sp.; GER1, GDP-4"-keto,6"-deoxy-d-mannose 3",5"-epimerase/4"-reductase of A. thaliana; GFS, GDP-l-Fuc synthetase of E. coli; TGD, TDP-d-glucose 4",6"-dehydratase of E. coli; GALE, UDP-d-glucose 4"-epimerase of E. coli. Positions on the ruler: 31–41, dinucleotide binding site; 162, 196, and 200, conserved S (T), Y, and K residues, respectively, of the catalytic triad of the short-chain dehydrogenase/reductase (SDR) protein family. *, conserved residues.