The tryptophan (trp) operon of Escherichia coli has become the basic reference structure for studies on tryptophan metabolism. Within the past five years the application of recombinant DNA and sequencing methodologies has permitted the characterization of the structural and functional elements in this gene cluster at the molecular level. In this summary report we present the complete nucleotide sequence for the five structural genes of the trp operon of E. coli together with the internal and flanking regions of regulatory information.

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.

In Escherichia coli, p-aminobenzoate (PABA) is synthesized from chorismate and glutamine in two steps. Aminodeoxychorismate synthase components I and II, encoded by pabB and pabA, respectively, convert chorismate and glutamine to 4-amino-4-deoxychorismate (ADC) and glutamate, respectively. ADC lyase, encoded by pabC, converts ADC to PABA and pyruvate. We reported that pabC had been cloned and mapped to 25 min on the E. coli chromosome (J. M. Green and B. P. Nichols, J. Biol. Chem. 266:12971-12975, 1991). Here we report the nucleotide sequence of pabC, including a portion of a sequence of a downstream open reading frame that may be cotranscribed with pabC. A disruption of pabC was constructed and transferred to the chromosome, and the pabC mutant strain required PABA for growth. The deduced amino acid sequence of ADC lyase is similar to those of Bacillus subtilis PabC and a number of amino acid transaminases. Aminodeoxychorismate lyase purified from a strain harboring an overproducing plasmid was shown to contain pyridoxal phosphate as a cofactor. This finding explains the similarity to the transaminases, which also contain pyridoxal phosphate. Expression studies revealed the size of the pabC gene product to be approximately 30 kDa, in agreement with that predicted by the nucleotide sequence data and approximately half the native molecular mass, suggesting that the native enzyme is dimeric.

SummaryIn plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a b-lyase that converts 4-amino-4-deoxychorismate (ADC) -the reaction product of the PabA and PabB enzymes -to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.

In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate. 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase. These two enzymes are encoded by ubiC and ubi4, respectively, and have been reported to map near one another at 92 min on the E. coli chromosome. We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubi4. The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubi4. ubiC was localized by subcloning, and overproducing plasmids were constructed. Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region. Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychorismate -. 4-aminobenzoate), the product of E. coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain. Of the several enzymes that convert chorismate to intermediates of E. coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized.Chorismate is the branch point precursor for the synthesis of many aromatic compounds in Escherichia coli. The seven major end products of chorismate anabolism are phenylalanine, tyrosine, tryptophan, ubiquinone, 4-aminobenzoate, menaquinone, and enterobactin. Chorismate itself undergoes five different conversions that result in those seven products. The tyrosine and phenylalanine pathways diverge following the isomerization of chorismate to prephenate, but two distinct, homologous chorismate mutase activities channel chorismate toward either phenylalanine or tyrosine (10). The menaquinone and enterobactin pathways diverge following the conversion of chorismate to 2-hydroxy-4-deoxychorismate (isochorismate) by isochorismate synthase (28,30). For 4-aminobenzoate synthesis, chorismate is first aminated to form 4-amino-4-deoxychorismate, which then undergoes ,-elimination of the enol-pyruvyl moiety to form 4-aminobenzoate (1,8,21,29). The first step in ubiquinone biosynthesis is the conversion of chorismate to 4-hydroxybenzoate by chorismate lyase (5, 15), in a reaction apparently similar to the second step of 4-aminobenzoate synthesis, catalyzed by 4-amino-4-deoxychorismate lyase. Finally, the first step in tryptophan synthesis is chorismate amination and p-elimination of the enol-pyruvyl group to form anthranilate, or 2-aminobenzoate (2).With the exception of the genes involved in ubiquinone biosynthesis, all of the E. coli genes encoding enzymes that use chorismate or 4-amino-4-deoxychorismate as substrates have been cloned and sequenced. These include pheA (chorismate mutase-prephenate dehydratase), tyrA (chorismate mutase-prephenate dehydrogenase) (10), entC (isochorismate synth...

The complete nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli were aetermined. 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. In several studies it has been observed that evolutionary changes in the nucleotide sequences of structural genes have accumulated to an extent greater than expected from amino acid sequence variation (1-4). These observations have stimulated an interest in the role of natural selection in nucleotide sequence variation and have raised the question: What fraction of such variation is due to nonselective or neutral nucleotide changes (5-8)? A comparable question has also been asked of protein primary sequence variation and, although our knowledge of protein structure and its relationship to function permits an appreciation of the participation of natural selection in protein structure evolution, as yet it has not been possible to ascertain what fraction of amino acid changes in homologous proteins are selectively neutral. Defining selectively important features of nucleotide sequences has proved equally difficult. This uncertainty perhaps explains why the nucleotide sequence divergence in the 3-globin genes of rabbits and humans has been cited as evidence for both neutral and selected nucleotide changes during the course of evolution (4,(8)(9)(10).In this report, we compare the nucleotide sequences of trpA of Escherichia coli and Salmonella typhimurnum. The products of these genes, the a-chain subunits of tryptophan synthetase, though divergent in both amino acid and nucleotide sequence (2, 11), are interchangeable without loss of function both in vitro (12-14) and in vivo (13,15). Furthermore, interspecies recombinants between trpAs of E. coli and S. typhimurium have been produced and, although the hybrid tryptophan synthetase a polypeptides differ appreciably in amino acid sequence from either parent, there is no apparent functional difference (16). We have determined the nucleotide sequence of trpA of these two organisms and can now directly evaluate the extent and type of synonymous nucleotide substitutions that have become established in this gene since the divergence of these two species.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. MATERIALS AND METHODSPlasmid JR42, a mini ColEl derivative carrying E. coli K-12 trpA, was the gift of Jorg Reif. The plasmid was prepared from E....

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Hybrid tryptophan synthetase at and .3 polypeptides were produced by genetic .recombination between the trpB-trpA regions ofEscherichia 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 a polypeptide despite many amino acid differences from one or the other of-the parental polypeptides. The five hybrid tryptophan synthetase a 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 gene trpA ofEscherichia coli and Salmonella typhimurium encodes the a polypeptide subunit of the a2f82 tryptophan synthetase complex. Comparison ofthe nucleotide sequences ofthe trpAs of the two species reveals that there are 199 sequence differences in the 804 nucleotide pairs in this gene (1). The corresponding a polypeptides have 40 amino acid differences in their 268 residues. Thus, most of the nucleotide differences between the two trpAs are in synonymous codons. Despite the 40 amino acid differences between the two a subunits, the polypeptides are functionally interchangeable, both in vitro (2-4) and in vivo (3,5). In this and comparable examples, the question arises as to whether the nucleotide and amino acid sequence differences that are seen in homologous genes and proteins are selectively favored in their respective organisms.The functional significance of the amino acid differences in the two tryptophan synthetase a subunits can be assessed by constructing hybrid trpAs containing sequences from both species and examining the properties of the corresponding hybrid polypeptides. By using this approach, one class of hybrid tryptophan synthetase a polypeptides was isolated and studied (6). Unfortunately, such recombinants were extremely rare and were obtained only when trpA' recombinants were selected in crosses between trpA mutants of the two species. The single class of hybrid a subunits examined resembled the parental polypeptides in all the functional tests that were performed.In this investigation, the difficulty ofobtaining hybrid a sub--units was overcome by incorporating inactive trpB-trpA segments of the two species into compatible multicopy plasmids and introducing both plasmids into bacteria with the trpB-trpA region deleted. The two plasmids were constructed so that the region of nucleotide sequence homology was confined to the trpB-trpA segment and so that genetic recombination in this segment could reconstitute intact trpB and trpA. Using this approach, we readily recovered hybrid trpBs and trpAs containing varying segments from the parental genes. It was also possible to modify the selection procedure slightly so that we could dete...