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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 nmol.g(-1) fresh weight.

Hybrid tryptophan synthetase alpha and beta polypeptides were produced by genetic recombination between the trpB--trpA regions of Escherichia coli and Salmonella typhimurium contained on compatible, multicopy plasmids. Intragenic recombination was decreased but still evident in recA cells. Genetic exchange occurred at many sites within trpA, but every recombinant gene produced a functional alpha polypeptide despite many amino acid differences from one or the other of the parental polypeptides. The five hybrid tryptophan synthetase alpha subunits examined resembled the parental polypeptides in catalytic function but differed in thermostability. The stability differences suggest that, as amino acid changes occurred in these proteins during the course of evolution, subsequent changes were limited to those that would allow retention of a desired protein conformation.

The complete nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli were determined. The nucleotide sequences are 24.8% divergent, compared with amino acid sequence divergence of 14.9%. Over half of the codons of each gene contain synonymous nucleotide changes. The pattern of synonymous nucleotide changes is consistent with the interpretation that such changes result from random mutational events. We do not find any evidence indicating that codon selection or RNA structure is of major selective value. We conclude that polypeptide function is the primary basis of selection in trpA and that most synonymous codon changes are selectively neutral.