FOLATE SYNTHESIS IN PLANTS: THE P-AMINOBENZOATE BRANCH IS INITIATED BY A BIFUNCTIONAL PABA-PABB PROTEIN THAT IS TARGETED TO PLASTIDS

Authors: BASSET, GILLES - UNIVERSITY OF FLORIDA; QUINLIVAN, EOIN - UNIVERSITY OF FLORIDA; RAVANEL, STEPHANE - UNIV JOSEPH FOURIER; NIOCHOLS, BRIAN - UNIVERSITY OF ILLINOIS; SHINOZAKI, KAZUO - RIKEN TSUKUBA INSTITUE; SEKI, MATOAKI - RIKEN TSUKUBA INSTITUTE; Giovannoni, James; ADAMS-PHILLIPS, LORI - BOYCE THOMPSON INSTITUTE; GREGORY, JESSE - UNIVERSITY OF FLORIDA; HANSON, ANDREW - UNIVERSITY OF FLORIDA

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2003
Publication Date: 2/10/2004
Citation: Basset, G., Quinlivan, E., Ravanel, S., Niochols, B., Shinozaki, K., Seki, M., Giovannoni, J.J., Adams-Phillips, L., Gregory, J., Hanson, A. 2004. Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional paba-pabb protein that is targeted to plastids. Proceedings of the National Academy of Sciences. 101:1496-1501.

Interpretive Summary: Tetrahydrofolate and its derivatives, collectively termed folates, are essential cofactors for one-carbon transfer reactions and thus are required for synthesis of methionine, purines, and thymidylate, and for interconversion of glycine and serine. Plants, fungi, and many microorganisms synthesize folates de novo, but humans and other higher animals do not and therefore require a dietary supply. As folate deficiency is a worldwide health problem and plant foods are major sources of folates for humans, there is much interest in engineering plants to enhance folate content. To do this, the plant folate synthesis pathway must first be understood. It is not known how plants synthesize the p-aminobenzoate (pABA) moiety of folates. In this study, we identified tomato and Arabidopsis cDNAs encoding hybrid PabA-PabB proteins and showed these proteins to be ADC synthases that are plastid-targeted. Because tomato fruit is our system for folate engineering and nothing was known about pABA synthesis in fruits, we also analyzed ADC synthase expression in relation to pABA levels during tomato fruit ripening.

Technical 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 aminodeoxychorismate (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 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 green fluorescent protein and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that pABA synthesis is plastidial. In the pericarp of tomato fruit, the level of PabA-PabB mRNA fell drastically as ripening advanced but there was no fall in total pABA content, which stayed between 0.7 and 2.3 nmol g-1 fresh weight.