3 Deoxy D arabino heptulosonic acid 7 phosphate synthase mutants of Salmonella typhimurium

Abstract

The first committed step of aromatic animo acid biosynthesis in S. typhimurium was shown to be catalyzed by 3 isoenzymes of 3 deoxy D arabino heptulosonic acid 7 phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine and tyrosine specific branch point enzymes, respectively. The phenylalanine inhibitable isoenzyme was the predominant DAHP synthase in wild type cells, and only the tyrosine inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine inhibitable isoenzyme. No cross inhibition of either the tyrosine or phenylalanine inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan inhibitable isoenzyme was partially purified from extracts of a strain lacking the other 2 isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4 phosphate, or an aldehydic compound resulting from degradation of erythrose 4 phosphate by periodate.