Structural Studies on a Mitochondrial Glyoxalase II

Authors

Gishanthi P. K. Marasinghe, Miami University - Oxford
Ian M. Sander, Miami University - Oxford
Brian Bennett, Marquette UniversityFollow
Gopal R. Periyannan, Eastern Illinois UniversityFollow
Ke-Wu Yang, Miami University - OxfordFollow
Christopher A. Makaroff, Miami University - Oxford
Michael W. Crowder, Miami University - Oxford

Document Type

Article

Language

eng

Format of Original

8 p.

Publication Date

12-9-2005

Publisher

American Society for Biochemistry and Molecular Biology

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Original Item ID

doi: 10.1074/jbc.M509748200

Abstract

Glyoxalase 2 is a β-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and ¹H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ± 0.15 eq of iron and 1.31 ± 0.05 eq of Zn(II) and to exhibit k_cat and K_m values of 129 ± 10 s^-1 and 391 ± 48 μm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. ¹H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-Å resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).

Comments

Published version. Journal of Biological Chemistry, Vol. 280, No. 49 (December 9, 2005): 40668-40675. DOI. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Used with permission.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

Recommended Citation

Marasinghe, Gishanthi P. K.; Sander, Ian M.; Bennett, Brian; Periyannan, Gopal R.; Yang, Ke-Wu; Makaroff, Christopher A.; and Crowder, Michael W., "Structural Studies on a Mitochondrial Glyoxalase II" (2005). Physics Faculty Research and Publications. 79.
https://epublications.marquette.edu/physics_fac/79