CYCLOHEXADIENYL DEHYDRATASE FROM PSEUDOMONAS-AERUGINOSA IS A PERIPLASMIC PROTEIN

The gene encoding cyclohexadienyl dehydratase from Pseudomonas aerugin osa, designated pheC, was cloned in Escherichia coli and sequenced rec ently by Zhao et al. (journal of Biological Chemistry 267, 2487-2493, 1992). N-Terminal sequencing of the purified cyclohexadienyl dehydrata se yielded a run of 11 residues which matched perfectly with the deduc ed amino acid residues 26-36. This showed that a 25 residue peptide wa s cleaved from the N-terminus of a preprotein formed in E. coli. The a mino acid composition of the 25 residue peptide was typical of signal sequences for periplasmic proteins. Most or all of the cyclohexadienyl dehydratase was released from P. aeruginosa and E. coli carrying the pheC gene following spheroplast formation, osmotic shock or chloroform treatment. The location of the enzyme in the periplasm of both E. col i and P. aeruginosa was confirmed by Western blotting analysis using a ntibody prepared against PheC. Electron microscopy using immunogold la belling showed an apparent localization of cyclohexadienyl dehydratase at the polar regions of the periplasmic space in E. coli.