Structural Variation in Bacterial Glyoxalase I Enzymes

Abstract

The glyoxalase system catalyzes the conversion of toxic, metabolically produced ␣-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn 2؉ and non-Zn 2؉ activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Grampositive bacteria that has been fully characterized as to its threedimensional structure and its detailed metal specificity. It is a Ni 2؉ /Co 2؉ -activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn 2؉ -bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements. Methylglyoxal (MG),3 an ␣-ketoaldehyde produced from a number of different enzymes and pathways, including triosephosphate isomerase, amino acid degradation, and acetoneconverting monooxygenases, is found in both prokaryotic and eukaryotic organisms (1-4). MG is a cytotoxic compound that can be produced to a level as high as 0.4 mM per cell per day, leading to protein synthesis inhibition, adduct formation with proteins, DNA, and RNA and can promote advanced glycation end products (5-10).A major contributing pathway involved in the detoxification of MG is the glyoxalase system. This two-enzyme system consists of glyoxalase I (GlxI) and glyoxalase II (GlxII) that convert ␣-ketoaldehydes into their corresponding 2-hydroxycarboxylic acids (D-lactate in the case of methylglyoxal), using an intracellular thiol as a cofactor/cosubstrate (Fig. 1). The first enzyme, GlxI (S-D-lactoylglutathione methylglyoxal lyase (isomerizing), EC 4.4.1.5), converts a hemithioacetal, the product of the nonenzymatic reaction between MG and a thiol, such as glutathione (GSH), to S-D-lactoylglutathione. GlxI is a metalloenzyme that can be divided into two classes, Zn 2ϩ activation (i.e. Homo sapiens GlxI (11)) and non-Zn 2ϩ activation (being selectively Ni 2ϩ /Co 2ϩ -activated, i.e. Escherichia coli GlxI (12)). It is likely that almost all Gram-negative prokaryotes possess Ni 2ϩ /Co 2ϩ -activated GlxI with the...

Nonredox Nickel Enzymes

“…Crystal structures of class I enzymes (mouse, PDB code 2ZA0; 117 and human, 118 with S -( N -hydroxy- N - p -iodophenylcarbamoyl)glutathione, 1QIN, with S - p -nitrobenzy-loxycarbonylglutathione, 1QIP, with benzylglutathione, 1FRO, with an N -hydroxypyridone inhibitor, 3VW9, and a Q33E/ E172Q double mutant, 1BH5) and class II enzymes ( L. major , 2C21; 99a L. infantum; 119 E. coli , 120 apo, 1FA8; with Ni(II) bound, 1F9Z; with Co(II) bound, 1FA6; with Zn(II) bound, 1FA5; and with Cd(II) bound, 1FA7; C. acetobutylicum , 114 with Zn(II) bound, 2QH0, or Ni(II) bound, 3HDP) are available. Comparisons of the structures show that both classes of Glo I are members of the βαβββ protein structure superfamily and are homodimers, or in some cases fused dimers (e.g., S. cerevisiae 121 and P. falciparum 122 ), composed of monomer units that have two structurally equivalent domains that provide one Glu and one His involved in binding the metal at the dimer interface (Figure 12).…”

“…The relationship between coordination geometry and activity is further supported by the structural data from Glo I isolated from C. acetobutylicum that has a different quaternary structure such that the ligands forming the active sites are derived from the same polypeptide chain. 114 Nonetheless, it has the shorter amino acid sequence typical of the bacterial enzymes and is activated by Ni(II), but not by Zn(II) ions, and therefore belongs to class II. The structure of the Ni(II) complex reveals that it is six-coordinate with a structure similar to that of the E. coli enzyme, and a five-coordinate Zn(II) complex with one aqua ligand.…”

“…The structure of the Ni(II) complex reveals that it is six-coordinate with a structure similar to that of the E. coli enzyme, and a five-coordinate Zn(II) complex with one aqua ligand. 114 …”

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes

“…However, both the active sites in the homodimeric GLYI from Clostridium acetobutylicum are located within the single subunits [73]. Similarly, even GLYI from Pseudomonas putida forms an active site within the same polypeptide in its monomeric form [60].…”

Synthesis of azide derivative and discovery of glyoxalase pathway inhibitor against pathogenic bacteria

“…Recently, GlxI enzyme is reported in gram positive bacteria - Clostridium acetobutylicum . 12 Interestingly, the evidence for the functional protein has been well determined for the same microorganism. Most importantly, the same glyoxal pathway is not found in human cells.…”