Analysis of growth and activity of transformed Escherichia coli cells containing the Entamoeba histolytica enzyme EhADH2 by using metal ions and chelators to confirm the presence of an iron active center within the ADH domain

CHED 931

Melissa J. Roberto, mroberto100@hawks.rwu.edu and Dr. Avelina Espinosa, aespinosa@rwu.edu. Department of Biology, Roger Williams University, One Old Ferry Rd., Bristol, RI 02809
Fifty million people a year suffer from amoebiasis, caused by the intestinal protozoan Entamoeba histolytica. Studies show that the iron active center of the ADH domain in the EhADH2 enzyme from E. histolytica is essential for fermentative growth. This study demonstrates the importance of iron (II) in anaerobic growth of transformed E. coli cells and the biochemical activity of EhADH2. Transformed E. coli cells were grown under optimal anaerobic growth conditions. The optimal medial growth conditions were altered in increased ferrous (II), in zinc (II) (a competitive inhibitor), or in 1, 10- phenanthroline (a metal chelator). The E. coli cells grown under the various conditions were lysed by French press, and the enzyme was purified by FPLC. The isolated enzyme concentration was determined using the Bradford Assay. Activity of the EhADH2 enzyme was determined with substrate kinetics using acetaldehyde as a substrate and monitored by UV/Vis (340 nm). NIH-NCRR Grant #2P20RR1645704