Folate synthesis in plants: The p -aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids

Abstract

It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates. In Escherichia coli, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and Plasmodium spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called ''PABA synthases,'' although it is unclear whether they produce PABA or ADC. Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an E. coli pabA pabB double mutant and a yeast PABA-synthase deletant. The partially purified recombinant Arabidopsis protein did not produce PABA unless the E. coli PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol⅐g ؊1 fresh weight.

Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis

“…Because the total PABA pool in nonengineered tomatoes is about the same size (1-2 nmol per g of FW) (14,21) as the folate increase in GCHI ϩ fruit, we examined the relationship between the levels of folate and PABA. A significant negative correlation was evident, and the GCHI ϩ fruit with the highest folate contents had very little PABA (Fig.…”

“…The validity of this approach has been demonstrated in food-grade lactic acid bacteria, in which engineering tripled folate production (10). Because the plant folate synthesis pathway is now known (2,(11)(12)(13)(14)(15), folate engineering can be attempted with plants, and several groups have begun doing this (16)(17)(18)(19). We have chosen to engineer the folate pathway in tomatoes, in view of their worldwide importance as a food crop, their well developed molecular genetics, and their relatively low folate content (3,13).…”

“…In plants, the pteridine moiety is produced in the cytosol, PABA is formed in plastids, and the two are coupled together and glutamylated in mitochondria (Fig. 1B) (11)(12)(13)(14)(15). In tomato fruit, the first enzyme of pteridine synthesis, GTP cyclohydrolase I (GCHI), disappears at the onset of ripening (13), cutting off the pteridine supply.…”

“…In tomato fruit, the first enzyme of pteridine synthesis, GTP cyclohydrolase I (GCHI), disappears at the onset of ripening (13), cutting off the pteridine supply. As ripening tomatoes contain large pools of glutamate (20) and moderate pools of PABA (14,21), a rational gambit to enhance folate synthesis is thus to engineer sustained GCHI activity (13). An attractive way to achieve sustained GCHI activity is to use a synthetic gene based on mammalian GCHI because this enzyme is unlikely to be subject to retroinhibition in planta.…”

Folate Polyglutamylation is Required for Rice Seed Development

“…The reduction in monoglutamated folates may be a result of folate breakdown, as polyglutamylated protein-bound folates are less susceptible to oxidative breakdown (Suh et al 2001). All ten steps involved in the plant folate biosynthesis pathway have been described and characterised, and the expression of several enzymes involved in this pathway has been correlated with the folate pool and the carbon-one unit demands in various physiological situations (Basset et al, 2002(Basset et al, , 2004a(Basset et al, , 2004bJabrin et al, 2003). Indeed, this study has confirmed these previous observations, in that the reduction in folate abundance in tissues of the fpgs03g knockout line was shown to be correlated with an increased expression of a number of folate biosynthesis genes, namely GTPCHI, ADC synthase, ADC lyase and HPPK/DHPS compared to wild type (Fig.…”