Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.     

Chemical modification of PABA synthase.

p-Aminobenzoic acid (PABA) synthase catalyses the first step in folic acid biosynthesis, the conversion of chorismate to p-aminobenzoate. In general, difficulties in purification have permitted only limited investigation of this enzyme. However, in an attempt to identify possible active site residues, the E. coli enzyme has been incubated with a range of protein modifying agents. Results indicate that cysteine, histidine, arginine and tyrosine residues are important for enzyme activity. Attempts were made to determine the subunits upon which these residues were located.     

Interaction between D-amino acid oxidase and small molecules.

1. From the standpoint of monomer-dimer equilibrium of hog kidney D-amino acid oxidase [EC 1.4.3.3] and the interaction between the enzyme and small molecules, the effect of pH on the binding of p-aminobenzoate to the monomer and dimer of the enzyme was studied by kinetic methods and spectrophotometric titration. 2. The maximum binding number of p-aminobenzoate to the dimer is two molecules, and there is no interaction between the two active sites of the dimer (i.e., no cooperativity) over the range of pH from 6.5 to 10. 3. The affinity of the dimer for p-aminobenzoate is several times higher than that of the monomer at pH 6.5-10, and consequently p-aminobenzoate induces dimerization in the equilibrium state of D-amino acid oxidase. The interaction energy of two subunits of the dimer is stabilized by the binding of p-aminobenzoate by 1-2 kcal/mole over the pH range studied. 4. The binding sites of the quasi-substrate, p-aminobenzoate, in the dimer and the intersubunit binding site of the dimer are clearly different, because p-aminobenzoate induces dimerization of the enzyme. 5. The pK values of ionizing groups in the free monomer and the free dimer which participate in the binding of the competitive inhibitor, p-aminobenzoate, are approximately the same, 8.7, as determined from the pH dependence of the affinity of the inhibitor for the enzyme. Furthermore, no pK for the enzyme-inhibitor complex in the pH range 6.5-10 was observed. 6. There is no interaction between the two ionizing groups of the dimer during protonation-deprotonation, because a theoretical equation involving no cooperativity between the two ionizing groups in the dimer explains the results well.     

Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase.

Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related. Both enzymes are composed of two nonidentical subunits. Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB. These two enzymes catalyze similar reactions and produce similar products. The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes. Here we present the nucleotide sequence of pabB and compare it with that of trpE. Similarities are 26% at the amino acid level and 40% at the nucleotide level. We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase.     

Expression of Escherichia coli pabA.

Escherichia coli pabA encodes the glutamine amidotransferase subunit of p-aminobenzoate synthase. p-Aminobenzoate synthase catalyzes the conversion of chorismate and glutamine to 4-amino-4-deoxychorismate, which is then converted to p-aminobenzoate by a 4-amino-4-deoxychorismate lyase. The 5'-terminal segment of pabA was previously shown to be transcribed from two different promoters, one near the pabA coding sequence (P1) and one preceding fic (P2). However, a pabA-lacZ translational fusion was expressed only from the mRNA originating at P1. We have determined that expression of a pabA-lacZ chromosomal fusion is not changed by p-aminobenzoate limitation, growth rate, catabolite repression, overexpression of either p-aminobenzoate synthase subunit, or gene dosage of pabA and pabB. The lack of pabA expression from P2 appears to be the result of a stable secondary structure in the intergenic space preceding pabA that sequesters the pabA ribosome binding site. Disruption of the secondary structure by mutation allowed expression of pabA from P2, as did translation of ribosomes into the fic-pabA intergenic region.     

Origin of p-aminobenzoic acid from chorismic rather than iso-chorismic acid in Enterobacter aerogenes and Streptomyces species

Enzyme extracts from Enterobacter aerogenes (62-1), Streptomyces aminophilus, and Streptomyces coelicolor were used to investigate the biosynthesis of p-aminobenzoic acid. The enzyme preparations from E. aerogenes and S. aminophilus contained both p-aminobenzoate synthase and iso-chorismate synthase activity, and were able to convert both chorismic and iso-chorismic acid to p-aminobenzoic acid. The apparent KM for chorismic acid was, however, significantly lower than that for iso-chorismic acid, while the Vmax was identical for both substrates in both enzyme systems. The enzyme preparations from S. coelicolor did not contain iso-chorismate synthase activity and p-aminobenzoic acid synthesis took place in this system from chorismic acid only. It is concluded that iso-chorismic acid is not an obligatory intermediate in p-aminobenzoic acid biosynthesis in these organisms.     

Structure of Escherichia coli aminodeoxychorismate synthase: architectural conservation and diversity in chorismate-utilizing enzymes

Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.     

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.     

Subunit structure of anthranilate synthase from Neurospora crassa: Preparation and characterization of a protease-free form

The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of M_r 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.     

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene.

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.     

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.     

Flavanone synthase from Petroselinum hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue.

Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77 000 for the enzyme and about 42 000 for the subunits were determined respectively by sedimentation-equilibrium measurements and disc-gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrifugation and gel electrophoresis under partially denaturing conditions. Values of about 17 S and Mr = 0.62 X 10(6) were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical. Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, beta-alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3-oxoacyl-(acyl carrier protein) synthase is discussed.     

Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae: combinatorial expression of the five PRS genes in Escherichia coli

The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on Pi, to be temperature-sensitive, and inhibited by ADP.     

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F₁F₀-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.     

Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans.

Sixty independent tryptophan auxotrophs of Pseudomonas acidovorans were isolated and characterized for nutritional response to intermediates of the pathway, accumulation of intermediates, and levels of tryptophan-synthetic enzymes. Mutants for each of the seven proteins catalyzing the five steps of tryptophan synthesis were obtained. Transductional analysis established three unlinked chromosomal regions: trpE, trpGDC, and trpFBA. The order of the genes within the two clusters was not determined. The levels and enzymatic activities of wild-type and mutant strains indicated that trpE and trpGDC were repressed by tryptophan. In contrast, trpFBA was not derepressed significantly by starvation for tryptophan. The trpG mutants had an additional requirement for p-aminobenzoate, which suggested that anthranilate synthase subunit II also served as glutamine-binding protein in the analogous reaction catalyzed by p-aminobenzoate synthase. In addition, trpD mutants revealed the ability of P. acidovorans to degrade anthranilate via the beta-ketoadipate pathway.     

Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase

pABA (p-aminobenzoate) is a precursor of folates and, besides esterification to glucose, has no other known metabolic fate in plants. It is synthesized in two steps from chorismate and glutamine, the first step being their conversion into glutamate and ADC (4-aminodeoxychorismate). In Escherichia coli, two proteins forming a heterodimeric complex are required for this reaction, but, in plants and lower eukaryotes, a single protein is involved. The Arabidopsis enzyme was expressed in E. coli and was purified to homogeneity. The monomeric enzyme (95 kDa) catalyses two reactions: release of NH3 from glutamine (glutaminase activity) and substitution of NH3 for the hydroxy group at position 4 of chorismate (ADC synthase activity). The kinetic parameters of the plant enzyme are broadly similar to those of the bacterial complex, with K(m) values for glutamine and chorismate of 600 and 1.5 microM respectively. As with the bacterial enzyme, externally added NH3 was a very poor substrate for the plant enzyme, suggesting that NH3 released from glutamine is preferentially channelled to chorismate. The glutaminase activity could operate alone, but the presence of chorismate increased the efficiency of the reaction 10-fold, showing the interdependency of the two domains. The plant enzyme was inhibited by dihydrofolate and its analogue methotrexate, a feature never reported for the prokaryotic system. These molecules were inhibitors of the glutaminase reaction, competitive with respect to glutamine (K(i) values of 10 and 1 microM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.     

The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits

It is now clearly established that dimerization of the F(1)F(o) ATP synthase takes place in the mitochondrial inner membrane. Interestingly, oligomerization of this enzyme seems to be involved in cristae morphogenesis. As they were able to form homodimers, subunits 4, e, and g have been proposed as potential ATP synthase dimerization subunits. In this paper, we provide evidence that subunit h, a peripheral stalk component, is located either at or near the ATP synthase dimerization interface. Subunit h homodimers were formed in mitochondria and were found to be associated to ATP synthase dimers. Moreover, homodimerization of subunit h and of subunit i turned out to be independent of subunits e and g, confirming the existence of an ATP synthase dimer in the mitochondrial inner membrane in the absence of subunits e and g. For the first time, this dimer has been observed by BN-PAGE. Finally, from these results we are now able to update our model for the supramolecular organization of the ATP synthase in the membrane and propose a role for subunits e and g, which stabilize the ATP synthase dimers and are involved in the oligomerization of the complex.     

The effects of acetate, metal cations, phenobarbitone, porphyrogens and substrates of glycine acyltransferase on the utilization of haem by rat liver apo-(tryptophan pyrrolase).

1. The utilization of haem by rat liver apo-(tryptophan pyrrolase) under basal conditions and after enhancement of the enzyme activity by various mechanisms was studied under the influence of treatments affecting various aspects of liver haem metabolism. 2. These treatments were: benzoate and p-aminobenzoate as substrates of glycine acyltransferase, acetate as an inhibitor of 5-aminolaevulinate synthase activity, enhancement of 5-aminolaevulinate dehydratase by aluminium, destruction of haem and inhibition of ferrochelatase by porphyrogens, increased haem utilization by phenobarbitone and enhancement of haem oxygenase activity by metal cations. 3. The results show that the haem saturation of the apoenzyme is sensitive to all these treatments. 4. The possible usefulness of tryptophan pyrrolase in studying the regulation of liver haem is suggested.     

Escherichia coli abg genes enable uptake and cleavage of the folate catabolite p-aminobenzoyl-glutamate

Escherichia coli AbgT was first identified as a structural protein enabling the growth of p-aminobenzoate auxotrophs on exogenous p-aminobenzoyl-glutamate (M. J. Hussein, J. M. Green, and B. P. Nichols, J. Bacteriol. 180:6260-6268, 1998). The abg region includes abgA, abgB, abgT, and ogt; these genes may be regulated by AbgR, a divergently transcribed LysR-type protein. Wild-type cells transformed with a high-copy-number plasmid encoding abgT demonstrate saturable uptake of p-aminobenzoyl-glutamate (K(T)=123 microM); control cells expressing vector demonstrate negligible uptake. The addition of metabolic poisons inhibited uptake of p-aminobenzoyl-glutamate, consistent with this process requiring energy. p-Aminobenzoyl-glutamate taken in by cells expressing large amounts of AbgT alone is not rapidly metabolized to a form that is trapped in the cell, as the addition of nonradioactive p-aminobenzoyl-glutamate to these cells results in a rapid loss of intracellular label. The addition of nonradioactive p-aminobenzoate has no effect. The abgA, abgB, and abgAB genes were cloned into the medium-copy-number plasmid pACYC184; p-aminobenzoate auxotrophs transformed with the clone encoding abgAB demonstrated enhanced ability to grow on low levels of p-aminobenzoyl-glutamate. When transformed with complementary plasmids encoding high-copy levels of abgT and medium-copy levels of abgAB, p-aminobenzoate auxotrophs grew on 50 nM p-aminobenzoyl-glutamate. Our data are consistent with a model of p-aminobenzoyl-glutamate utilization in which AbgT catalyzes transport of p-aminobenzoyl-glutamate, followed by cleavage to p-aminobenzoate by a protein composed of subunits encoded by abgA and abgB. While endogenous expression of these genes is very low under the conditions in which we performed our experiments, these genes may be induced by AbgR bound to an unknown molecule. The true physiological role of this region may be related to some molecule similar to p-aminobenzoyl-glutamate, such as a dipeptide.