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Full-Text Articles in Physics
Converting Glx2-1 Into An Active Glyoxalase Ii, Pattraranee Limphong, Nicole E. Adams, Matthew F. Rouhier, Ross M. Mckinney, Melissa Naylor, Brian Bennett, Christopher A. Makaroff, Michael W. Crowder
Converting Glx2-1 Into An Active Glyoxalase Ii, Pattraranee Limphong, Nicole E. Adams, Matthew F. Rouhier, Ross M. Mckinney, Melissa Naylor, Brian Bennett, Christopher A. Makaroff, Michael W. Crowder
Physics Faculty Research and Publications
Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level S-lactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited …
Human Glyoxalase Ii Contains An Fe(Ii)Zn(Ii) Center But Is Active As A Mononuclear Zn(Ii) Enzyme, Pattraranee Limphong, Ross M. Mckinney, Nicole E. Adams, Brian Bennett, Christopher A. Makaroff, Thusitha Gunasekera, Michael W. Crowder
Human Glyoxalase Ii Contains An Fe(Ii)Zn(Ii) Center But Is Active As A Mononuclear Zn(Ii) Enzyme, Pattraranee Limphong, Ross M. Mckinney, Nicole E. Adams, Brian Bennett, Christopher A. Makaroff, Thusitha Gunasekera, Michael W. Crowder
Physics Faculty Research and Publications
Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to …
Metal Content Of Metallo-Β-Lactamase L1 Is Determined By The Bioavailability Of Metal Ions, Zhenxin Hu, Thusitha S. Gunasekera, Lauren J. Spadafora, Brian Bennett, Michael W. Crowder
Metal Content Of Metallo-Β-Lactamase L1 Is Determined By The Bioavailability Of Metal Ions, Zhenxin Hu, Thusitha S. Gunasekera, Lauren J. Spadafora, Brian Bennett, Michael W. Crowder
Physics Faculty Research and Publications
In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized …