Overexpression and purification of ferric enterobactin esterase from Escherichia coli. Demonstration of enzymatic hydrolysis of enterobactin and its iron complex.
The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein. The ferric enterobactin esterase (Fes) enzyme was purified as a 43-kDa monomer by gel filtration chromatography. Purified Fes preparations were examined for esterase activity on enterobactin and its metal complexes and for iron reduction from ferric complexes of enterobactin and 1,3,5-tris(N,N',N"-2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), a structural analog lacking ester linkages. Fes effectively catalyzed the hydrolysis of both enterobactin and its ferric complex, exhibiting a 4-fold greater activity on the free ligand. It also cleaved the aluminum (III) complex at a rate similar to the ferric complex, suggesting that ester hydrolysis of the ligand backbone is independent of any reductive process associated with the bound metal. Ferrous iron was released from the enterobactin complex at a rate similar to ligand cleavage indicating that hydrolysis and iron reduction are tightly associated. However, no detectable release of ferrous iron from the MECAM complex implies that, with these in vitro preparations, metal reduction depends upon, and is subsequent to, the esterase activity of Fes. These observations are discussed in relation to studies which show that such enterobactin analogs can supply growth-promoting iron concentrations to E. coli.
Brickman
T J
TJ
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia 65212.
McIntosh
M A
MA
D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005784:Gene Amplification; D015870:Gene Expression; D006868:Hydrolysis; D010957:Plasmids; D011401:Promoter Regions, Genetic
D029968:Escherichia coli Proteins; D004758:Enterobactin; D002265:Carboxylic Ester Hydrolases; C075139:ferric enterobactin esterase, E coli