PEPTIDYL PROLYL CIS-TRANS-ISOMERASE FROM ESCHERICHIA-COLI - A PERIPLASMIC HOMOLOG OF CYCLOPHILIN THAT IS NOT INHIBITED BY CYCLOSPORINE-A

The prokaryotic peptidyl-prolyl cis-trans-isomerase called 'rotamase', a homolog of the human cyclophilin, has been identified in Escherichia coli. The E. coli rotamase, a product of the gene we suggest be called 'rot', has been purified to heterogeneity after cloning of the gene by the polymerase chain reaction and its overexpression in E. coli. Based on the chymotrypsin-coupled assay using the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the purified protein has rotamase activity identical to human cyclophilin with a catalytic efficiency close to the upper diffusional limit (k(cat)/K(m) ≃ 1.0 x 107 M-1·s-1 at 10°C). Unlike the human cyclophilins, however, the E. coli rotamase is not significantly inhibited by the immunosuppressant drug cyclosporin A. By spheroplast fractionation of cells harboring the expression vector for the complete rot gene, the rotamase is located in the periplasm, where it could function in refolding of secreted proteins.