Publications

• Deciphering the role of the type II glyoxalase isoenzyme YcbL (GlxII-2) in Escherichia coli.

  Reiger M., Lassak J., Jung K.

  In Escherichia coli, detoxification of methylglyoxal (MG) requires glyoxalases I and II. Glyoxalase I (gloA/GlxI) isomerizes the hemithioacetal, formed spontaneously from MG and glutathione (GSH) to S-lactoylglutathione (SLG), which is hydrolyzed by glyoxalase II (gloB/GlxII) to lactate and GSH. Y ... >> More

  In Escherichia coli, detoxification of methylglyoxal (MG) requires glyoxalases I and II. Glyoxalase I (gloA/GlxI) isomerizes the hemithioacetal, formed spontaneously from MG and glutathione (GSH) to S-lactoylglutathione (SLG), which is hydrolyzed by glyoxalase II (gloB/GlxII) to lactate and GSH. YcbL from Salmonella enterica serovar Typhimurium is an unusual type II glyoxalase whose role in MG detoxification has remained enigmatic. Here we show that YcbL (gloC/GlxII-2) acts as an accessory type II glyoxylase in E. coli. The two isoenzymes have additive effects and ensure maximal MG degradation. << Less

  FEMS Microbiol. Lett. 362:1-7(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural studies on a mitochondrial glyoxalase II.

  Marasinghe G.P., Sander I.M., Bennett B., Periyannan G., Yang K.W., Makaroff C.A., Crowder M.W.

  Glyoxalase 2 is a beta-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 ... >> More

  Glyoxalase 2 is a beta-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 (1)H 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 microm, 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. (1)H 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-A 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). << Less

  J. Biol. Chem. 280:40668-40675(2005) [PubMed] [EuropePMC]

• Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme.

  O'Young J., Sukdeo N., Honek J.F.

  Cytotoxic methylglyoxal is detoxified by the two-enzyme glyoxalase system. Glyoxalase I (GlxI) catalyzes conversion of non-enzymatically produced methylglyoxal-glutathione hemithioacetal into its corresponding thioester. Glyoxalase II (Glx II) hydrolyzes the thioester into d-lactate and free gluta ... >> More

  Cytotoxic methylglyoxal is detoxified by the two-enzyme glyoxalase system. Glyoxalase I (GlxI) catalyzes conversion of non-enzymatically produced methylglyoxal-glutathione hemithioacetal into its corresponding thioester. Glyoxalase II (Glx II) hydrolyzes the thioester into d-lactate and free glutathione. Glyoxalase I and II are metalloenzymes, which possess mononuclear and binuclear active sites, respectively. There are two distinct classes of GlxI; the first class is Zn2+-dependent and is composed of GlxI from mainly eukaryotic organisms and the second class is composed of non-Zn2+-dependent (but Ni2+ or Co2+-dependent) GlxI enzymes (mainly prokaryotic and leishmanial species). GlxII is typically Zn2+-activated, containing Zn2+ and either Fe3+/Fe2+ or Mn2+ at the active site depending upon the biological source. To address whether two classes of GlxII might exist, glyoxalase II from Escherichia coli was cloned and overexpressed and characterized. Unlike E. coli GlxI, which is non-Zn2+-dependent, Zn2+ activates the E. coli GlxII enzyme, with no evidence for Ni2+ metal utilization. << Less

  Arch. Biochem. Biophys. 459:20-26(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.

  Limphong P., McKinney R.M., Adams N.E., Makaroff C.A., Bennett B., Crowder M.W.

  In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-contai ... >> More

  In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)-Zn(II), and antiferromagnetically coupled Fe(II)-Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k(cat)/K(m), was invariant among the fractions. The value of k(cat)/K(m) governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k(cat)/K(m) on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites. << Less

  J. Biol. Inorg. Chem. 15:249-258(2010) [PubMed] [EuropePMC]

• Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis.

  Zang T.M., Hollman D.A., Crawford P.A., Crowder M.W., Makaroff C.A.

  Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and react ... >> More

  Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors. << Less

  J. Biol. Chem. 276:4788-4795(2001) [PubMed] [EuropePMC]