p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC.
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 pabA and pabB were assumed to be sufficient for de novo p-aminobenzoate biosynthesis. Recent studies, however, have shown that these proteins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely 4-amino-4-deoxychorismate. This intermediate is then converted to p-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 purification of aminodeoxychorismate lyase 4100-fold to near homogeneity. Further purification of this enzyme by high pressure liquid chromatography permitted isolation of a pure sample that yielded N-terminal sequence. A 64-fold redundant oligonucleotide probe was used to identify a lambda clone containing the gene encoding aminodeoxychorismate lyase. The aminodeoxychorismate lyase gene, designated pabC, was mapped to 25 min on the E. coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment. A strain harboring a pACYC184 recombinant containing pabC overproduced aminodeoxychorismate lyase activity 77-fold.
Green
J M
JM
Department of Biological Sciences, University of Illinois, Chicago 60680.
Nichols
B P
BP
D010129:4-Aminobenzoic Acid / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids
D015345:Oligonucleotide Probes; D007652:Oxo-Acid-Lyases; C066600:aminodeoxychorismate lyase; D010129:4-Aminobenzoic Acid