Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin

Abstract

Monogalactosyldiacylglycerol (MGDG) synthase (UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46) catalyzes formation of MGDG, a major structural lipid of chloroplast. We cloned a cDNA for the synthase from cucumber cDNA library. The full-length cDNA clone was 2142 bp, and it contains a 1575-bp open reading frame encoding 525 aa. The open reading frame consists of the regions for a mature protein (422 aa; Mr of 46,552) and transit peptide to chloroplast (103 aa). Although the molecular weight of mature protein region matched that purified from cucumber cotyledons, it was quite different from those purified from spinach (approximately 20 kDa) reported by other groups. The mature region of the protein was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The expression in E. coli showed that the protein catalyzed MGDG synthesis very efficiently. Therefore, we concluded that the cDNA encodes MGDG synthase in cucumber. In addition, the deduced amino acid sequence of the MGDG synthase cDNA showed homology with MurG of Bacillus subtilis and E. coli, which encode a glycosyltransferase catalyzing the last step of peptidoglycan synthesis in bacteria. This sequence homology implies that the machinery of chloroplast membrane biosynthesis is evolutionarily derived from that of cell wall biosynthesis in bacteria. This is consistent with the endosymbiotic hypothesis of chloroplast formation.

Figure 1

Nucleotide and deduced amino acid sequences of the cucumber MGDG synthase. Amino acid sequences of peptides obtained from the purified protein are underlined. Numbering begins at the first nucleotide of the sequence. The putative N terminus of the mature protein region is indicated by arrow. Open triangles indicate the 5′ and 3′ ends of the first clone obtained. Closed triangles indicate those of PCR products by 5′ RACE.

Figure 2

MGDG synthase expressed in E. coli. (a) Construction of plasmid with GST fusion expression vector of pGEX-3X to generate the mature region of MGDG synthase as a fusion protein with GST. (b) MGDG synthase activity measured by the formation of [³H]MGDG by cell-free extract of E. coli XL1-Blue transformed with pGEX-3X alone or pGEX-3X harboring the cDNA clone. We designated the expression vector pGEX-3X harboring the cDNA clone as pGEX-GT. GEX-3X and 1b-2 indicate E. coli XL1-Blue transformed with pGEX-3X and pGEX-GT, respectively. Plus (+) and minus (−) indicate the crude extracts from XL1-Blue grown in the presence or absence of IPTG, respectively.

Figure 3

TLC analysis of galactosylation products produced by the expressed fusion protein of MGDG synthase and GST in E. coli extracts. Lane 1, total lipids extracted from spinach, which were stained with anthrone–sulfuric acid; lanes 2 and 3, the reaction products of the extract from GEX-3X, which are untreated and treated with IPTG (1 mM final concentration), respectively; lanes 4 and 5, the reaction products of the extract from 1b-2, which are untreated and treated with IPTG, respectively; and lane 6, the reaction products of MGDG synthase purified from cucumber cotyledons. SQDG, sulfoquinovosyldiacylglycerol.

Figure 4

Alignment of the deduced amino acid sequence of MGDG synthase (mature region) with those of the MurG protein from B. subtilis (12) and E. coli (19, 20). Amino acids that are identical between MGDG synthase and the other proteins are highlighted in black. Four typical homologous regions (I, II, III, and IV) are indicated by underlining.