title: The binding of iron and zinc to glyoxalase II occurs exclusively as di-metal centers and is unique within the metallo-beta-lactamase family.
abstract: Cytosolic glyoxalase 2 (GLX2-2) from Arabidopsis thaliana is a metalloenzyme that has been shown to bind a mixture of Zn, Fe, or Mn when produced in cells grown in rich media. In an effort to prepare metal-enriched samples, GLX2-2 was over-expressed in minimal media containing either Zn, Fe, or Mn. The resulting enzymes bound an average of 1 equivalent of metal ion and were partially enriched with a specific metal ion. The enzymes produced in minimal media were active towards the substrate S-D-lactoylglutathione, yielding kcat/ Km values similar to those of rich media GLX2-2. EPR studies on minimal media GLX2-2 samples revealed spectra which were identical to those over-expressed in rich media that contained nearly 2 equivalents of metal. The EPR spectra showed the presence of antiferromagnetically and ferromagnetically coupled, dinuclear metal centers. EXAFS spectra on the minimal media GLX2-2 samples over-expressed in the presence of Fe or Zn were also very similar to those of the rich media GLX2-2 samples, indicating the presence of dinuclear metal centers. The EXAFS studies also demonstrate that Zn(II) and Fe (in the Fe-enriched sample) are distributed in the dinuclear site. These data indicate that the minimal media GLX2-2 samples are a mixture of fully loaded, dinuclear metal-containing enzyme and metal-free enzyme. This characteristic of A. thaliana GLX2-2 makes it unique among the other members of the metallo-beta-lactamase family in that it does not ever appear to exist as a mononuclear metal ion containing enzyme and that it exhibits positive cooperativity in metal binding.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 112 Hughes Hall, Oxford, OH 45056, USA.
authors.forename: Nathan F
authors.initials: NF
authors.lastname: Wenzel
authors.forename: Anne L
authors.initials: AL
authors.lastname: Carenbauer
authors.forename: Mary Pam
authors.initials: MP
authors.lastname: Pfiester
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.forename: Wolfram
authors.initials: W
authors.lastname: Meyer-Klaucke
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Makaroff
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013057:Spectrum Analysis; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D014965:X-Rays; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry; D000099301:Hydroxyacylglutathione Hydrolase
chemical_list: D029681:Arabidopsis Proteins; D005978:Glutathione; D007501:Iron; D013869:Thiolester Hydrolases; D015032:Zinc; D001618:beta-Lactamases; D000099301:Hydroxyacylglutathione Hydrolase; C013585:S-lactoylglutathione