Human Glx2 contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme
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.
[Show abstract] [Hide abstract] 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.0Comments 11Citations
- "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. "
[Show abstract] [Hide abstract] ABSTRACT: Glyoxalases are known to play a very important role in abiotic stress tolerance. This two-step pathway detoxifies ubiquitously present cytotoxic metabolite methylglyoxal, which otherwise increases to lethal concentrations under various stress conditions. Methylglyoxal initiates stress-induced signaling cascade via reactive oxygen species, resulting in the modifications of proteins involved in various signal transduction pathways, that eventually culminates in cell death or growth arrest. The associated mechanism of tolerance conferred by over-expression of methylglyoxal-detoxifying glyoxalase pathway mainly involves lowering of methylglyoxal levels, thereby reducing subsequently induced cellular toxicity. Apart from abiotic stresses, expression of glyoxalases is affected by a wide variety of other stimuli such as biotic, chemical and hormonal treatments. Additionally, alterations in cellular milieu during plant growth and development also affect expression of glyoxalases. The multiple stress-inducible nature of these enzymes suggests a vital role for glyoxalases, associating them with plant defense mechanisms. In this context, we have summarized available transcriptome, proteome and genetic engineering- based reports in order to highlight the involvement of glyoxalases as important components of plant stress response. The role of methylglyoxal as signaling molecule is also discussed. Further, we examine the suitability of glyoxalases and methylglyoxal as potential markers for stress tolerance.0Comments 6Citations
- "These metal ions are essential for the catalytic activity (Zang et al., 2001) and their binding occurs as di-nuclear metal ion centers (Wenzel et al., 2004 ). However , human GLYII is active as a mononuclear enzyme despite the presence of Fe(II)/Zn(II) di-nuclear center (Cameron, 1999; Limphong et al., 2009a). Plant glyoxalases have been largely studied from a physiological perspective, being linked to stress tolerance in plants. "
[Show abstract] [Hide abstract] 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.0Comments 2Citations
- "Glo I enzyme is found only in cytoplasmic space while Glo II enzyme is found in different cellular compartments (nucleus, endoplasmatic reticulum, and mitochondria). In eukaryotes multiple forms of Glo II are found in mitochondria (both in intermembrane space and in the matrix) while only one form of cytosolic Glo II exists, which resembles the intermembrane space form . 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. "