THE JOURNAL OF BIOLOGICAL CHEMISTRY zyxwvutsrqpon 8 1991 by The American Society for Biochemistry and Molecular Biology, Inc zyxwvutsrqpo Vol. 266, No. 20, Issue of July 15, pp. 12971-12975,1991 zy Printed zy in zyx U. S. A. p-Aminobenzoate Biosynthesis in zyxw Escherichia coli PURIFICATION OF AMINODEOXYCHORISMATE LYASE AND CLONING OF pabC* (Received for publication, February 5, 1991) Jacalyn M. Green andBrian P. Nichols$ From the Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois, Chicago, Illinois 60680 p-Aminobenzoate, a component of the vitamin folate, is one of seven compounds derived from the aromatic precursor chorismate in Escherichia coli. Historically the gene products of pabAand pabB were assumed to be sufficient for de novop-aminobenzoate biosynthesis. Recent studies, however, have shown that these pro- teins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely zyxwvuts 4-amino-4-deoxychorismate. This intermediate is then converted to p-aminobenzoate by a third dis- crete enzyme, aminodeoxychorismate lyase (Nichols, B. P., Seibold, A. S., and Doktor, S. Z. (1989) J. Biol. Chem. 264,8597-8601). Here we describe partial characterization of the intermediate and the purifica- tion of aminodeoxychorismate lyase 4100-fold to near homogeneity. Further purification of this enzyme by high pressure liquid chromatography permitted isola- tion of a pure sample that yielded N-terminal sequence. A 64-fold redundant oligonucleotide probe was used to identify a zyxwvutsr X clone containing the gene encoding ami- nodeoxychorismate lyase. The aminodeoxychorismate lyase gene, designatedpabc, was mapped to 2 5 min on the E. coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment. A strain harboring a pACYCl84 re- combinant containing pabC overproduced aminodeox- ychorismate lyase activity 77-fold. In Escherichia coli chorismic acid serves as the branch point precursor for metabolites essential in the biosynthesis of many important aromatic products. These metabolites and their end products include anthranilate (tryptophan), prephenate (tyrosine, phenylalanine), p-aminobenzoate (folic acid), iso- chorismate (enterochelin, menaquinone), and p-hydroxyben- zoate (ubiquinone). The reactions catalyzed by p-aminobenzoate synthase and anthranilate (0-aminobenzoate) synthase are strikingly simi- lar. In E. coli both enzymes are composed of nonidentical subunits. Component I (Mr zyxwvutsr - 50,000) of p-aminobenzoate synthase and anthranilate synthase is the gene product of pabB or trpE, respectively; component I1 (Mr - 20,000) of each enzyme is the gene product of pabA or trpG(D). Compo- nent I of either enzyme alone catalyzes an ammonia-depend- ent reaction, whereas inclusion of component I1 supports glutamine amidotransferase activity. Sequence data reveal * This work was supported in part by United States Public Health Service Grants GM44199 and A125106 from the National Institutes of Healthandinpart by theLaboratory for Molecular Biology, Department of Biological Sciences, the University of Illinois at Chi- cago. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. enough similarity between PabBandTrpE,and between PabA andTrpG, to suggest a common ancestor for both enzymes (Goncharoff and Nichols, 1984; Kaplan andNichols, 1983). Also, the chemistry of the reactions appears to be similar. It has been demonstrated that compound 1 (Scheme 1) is converted to anthranilate by component I of anthranilate synthase from Serratia marcescens (Teng and Ganem, 1984; Policastro et al., 1984). The analogous intermediate in p- aminobenzoate biosynthesis (compound 2, Scheme l), 4- amino-4-deoxychorismate, was also synthesized and shown to be kinetically competent with crude extract from E. coli (Teng et al., 1985). Preliminary experiments by Ye et al. (1990) also supportthisstructureas an intermediatein-ammonia-de- pendent p-aminobenzoate (PABA)’ biosynthesis from chor- ismate. Based on these data, the authors suggested that this third enzyme in PABA biosynthesis be called aminodeoxy- chorismate lyase, and in this report we use that name. Recently Nichols et al. (1989) showed that the conversion of chorismate to p-aminobenzoate occurs in two separate protein-catalyzed steps, not one, as previously thought. PabA and PabB act together to convert chorismate and glutamine to a diffusible intermediate, which is acted upon by a third protein to form p-aminobenzoate. This is in direct contrast to anthranilate synthase where no additionalproteinsare involved in the conversion of chorismate to anthranilate. In addition, the biosynthesis of anthranilate differs from that of PABA in that all the E. coli tryptophan biosynthetic genes are located in a single operon, whereas the two known E. coli p-aminobenzoate biosynthetic genes, pabA and pabB, are located at 74 and 40 min, respectively (Bachman, 1990). We are interested in investigating how PABA biosynthesis is regulated in the cell at all conceivable levels, the enzymatic, transcriptional, and translational. We describe here the fol- lowing: the purification of the third protein, aminodeoxychor- ismate lyase; the isolation and partial characterization of the physiological intermediate in the conversion of chorismate and glutamine to PABA and pyruvate; and the cloning and overexpression of pabC, the gene encoding aminodeoxychor- ismate lyase. MATERIALS AND METHODS Assay for PABA Synthesis-Assays were performed as described in Nichols et al. (1989). For routine assays of aminodeoxychorismate lyase, PabB and PabA were purified as described previously (Nichols et al., 1989). For HPLC experiments and studies designed to generate intermediate, PabA was additionally purified on a Mono P HR 5/20 FPLC column (Pharmacia LKB Biotechnology Inc.). Enzyme was loaded onto a column equilibrated with 25 mM imidazole, pH 7.4, 5% glycerol, and 10 mM 2-mercaptoethanol. Enzyme activity was eluted with 10-fold diluted polybuffer 74 (Pharmacia), pH 4.0, 5% glycerol, The abbreviations used are: PABA, p-aminobenzoic acid; amino- deoxychorismate, 4-amino-4-deoxychorismate; HPLC, high pressure liquid chromatography; FPLC, fast protein liquid chromatography. 12971 This is an Open Access article under the CC BY license.