| Our investigations have focused on the bacterial GlxI enzyme isolated from Escherichia coli. To extend our knowledge of glyoxalase I and the factors affecting this unexpected metal activation in E. coli GlxI, numerous kinetic analyses, structural studies, and sequence comparisons have been performed.; Examination of the kinetic parameters for E. coli GlxI indicated that the Km remains relatively constant in the presence of several catalytic metal ions. However, the activity of the enzyme is significantly altered. Maximal activity is observed in the Ni2+-reconstituted GlxI enzyme, with decreasing activity seen with the following metals; Co 2+, Mn2+, Fe2+, and Cd2+.; Extensive structural studies were performed on E. coli GlxI. The chemical shift from a 113Cd NMR study on Cd 2+-GlxI was consistent with oxygen and nitrogen ligands around the metal, as predicted based on sequence similarity to the H. sapiens GlxI enzyme. Electron paramagnetic resonance (EPR) analysis of Mn 2+-GlxI indicated an octahedral metal environment. X-ray absorption spectroscopy (XAS) on Ni2+-GlxI confirmed these findings. The metal ligands in the E. coli GlxI active site were identified as His5, Glu56, His74, and Glu122. Two water molecules complete the octahedral coordination around Ni2+, Co2+, and Cd 2+. Interestingly, in E. coli Zn2+-GlxI, the metal has only one water molecule and hence a trigonal bipyramidal coordination. It appears that an octahedral metal environment is required to produce an active enzyme.; The first metal ligand, His5, was changed by site-directed mutagenesis to a glutamine, the ligand found in the H. sapiens enzyme. Surprisingly, the metal affinity of this mutant E. coli GlxI enzyme was greatly decreased.; The sequence of E. coli GlxI was utilized to search the National Center for Biotechnology Information sequence databases. Twenty-eight putative GlxI sequences were identified, including nineteen from pathogenic organisms. Comparative analysis of these sequences revealed consistent alterations between the bacterial GlxI sequences and the H. sapiens GlxI sequence.; The DNA postulated to encode GlxI enzymes from Yersinia pestis and Pseudomonas aeruginosa were isolated and placed in overexpression vectors. Preliminary studies indicated these sequences do indeed encode enzymes with GlxI activity. Furthermore, addition of NiCl 2 but not ZnCl2 was observed to increase the activity of these enzymes. (Abstract shortened by UMI.)... |
