Human Glx2 contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme

Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
Biochemistry (Impact Factor: 3.02). 06/2009; 48(23):5426-34. DOI: 10.1021/bi9001375
Source: PubMed


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 the consensus Zn(2) site in Glx2 and that this site can also bind Co(II) and Ni(II), suggesting that Zn(II) binds to the consensus Zn(1) site. The NMR studies also reveal the presence of a dinuclear Co(II) center in Co(II)-substituted Glx2. Steady-state and pre-steady-state kinetic studies show that Glx2 containing only 1 equiv of Zn(II) is catalytically active and that the metal ion in the consensus Zn(2) site has little effect on catalytic activity. Taken together, these studies suggest that Glx2 contains a Fe(II)Zn(II) center in vivo but that the catalytic activity is due to Zn(II) in the Zn(1) site.

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    • "One of the rice GLY II, OsGLYII-3, has been well characterized in our laboratory (Yadav et al., 2007) and conferred enhanced salinity tolerance in rice (Singla-Pareek et al., 2008). Moreover, it was observed that AtGlx2-1 and OsGLYII-1 show activity other than GLY II (Limphong et al., 2009; Kaur et al., 2014c). Functional complementation of the yeast GLY II mutant by OsGLYII-2 (Figure 1) indicated that OsGLYII-2 is an active GLY II enzyme. "
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    ABSTRACT: Glyoxalase II (GLY II), the second enzyme of glyoxalase pathway that detoxifies cytotoxic metabolite methylglyoxal (MG), belongs to the super family of metallo-β-lactamases. Here, detailed analysis of one of the uncharacterized rice glyoxalase II family members, OsGLYII-2 was conducted in terms of its metal content, enzyme kinetics and stress tolerance potential. Functional complementation of yeast GLY II mutant (∆GLO2) and enzyme kinetics data suggested that OsGLYII-2 possesses characteristic GLY II activity using S-lactoylglutathione (SLG) as the substrate. Further, ICP-AES and modelled structure revealed that OsGLYII-2 contains a binuclear Zn/Fe centre in its active site and chelation studies indicated that these are essential for its activity. Interestingly, reconstitution of chelated enzyme with Zn2+, and/or Fe2+ could not reactivate the enzyme, while addition of Co2+ was able to do so. End product inhibition study provides insight into the kinetics of GLY II enzyme and assigns hitherto unknown function to reduced glutathione (GSH). Ectopic expression of OsGLYII-2 in E. coli and tobacco provides improved tolerance against salinity and dicarbonyl stress indicating towards its role in abiotic stress tolerance. Maintained levels of MG and GSH as well as better photosynthesis rate and reduced oxidative damage in transgenic plants under stress conditions seems to be the possible mechanism facilitating enhanced stress tolerance.This article is protected by copyright. All rights reserved.
    Full-text · Article · Jul 2014 · The Plant Journal
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    • "Glo II is a metal-dependent β-lactamase and shows a characteristic Zn 2 þ -binding motif, conserved in all known sequences, and a Zn(II) Fe(II) center [43] [44]. The biological meaning of the presence of Glo II and the absence of Glo I in mitochondria is still unclear. "
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    ABSTRACT: The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, protecting mitochondrial DNA against mitochondrial-derived ROS and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-Lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. We demonstrated SLG transport from cytosol to mitochondria by incubating substrates with radioactive compounds that showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. The incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria demonstrating transport not linked to ATP presence. Also mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data was confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II, the enzyme that catalyzes SLG hydrolysis, in inter-membrane space and mitochondrial matrix. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.
    Full-text · Article · Dec 2013 · Free Radical Biology and Medicine
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    • "The enzymes are glyoxalase I (Zn-containing, lactoylglutathione lyase; EC and II (Zn-containing , hydroxyacylglutathione hydrolase; EC and much is now known about their mechanisms of action [9] [10] [11]. The product of the pathway has been claimed to be D-lactic acid [12] [13], which leaves the cell and passes via the blood, putatively to the liver where it enters and is oxidized by peroxisomes [14], and possibly mitochondria [15], to pyruvate. "
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