L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant.

Abstract

Serine acetyltransferase (SAT) from Escherichia coli is subject to feedback inhibition by L-cysteine. A mutant was isolated which excretes L-cysteine because of a lesion in cysE, the structural gene for SAT, rendering the enzyme less feedback sensitive. To analyse the structural basis for this mutation the cysE genes both from wild-type E. coli and the mutant strain were cloned and their nucleotide sequences determined. The cysE gene contained an open reading frame consisting of 819 bp, equivalent to a protein of 273 amino acids. The mutant gene showed a single base change in position 767 resulting in a methionine to isoleucine substitution. A causal connection between this SAT sequence alteration, feedback insensitivity and L-cysteine excretion was demonstrated. The SAT from the wild-type strain was purified. It was composed of a single polypeptide chain migrating in SDS gels according to an Mr of 34,000. As in Salmonella typhimurium, the enzyme was associated in a bifunctional complex with O-acetylserine (thiol)-lyase.