Overproduction and characterization of a dimeric non-zinc glyoxalase I from Escherichia coli: evidence for optimal activation by nickel ions.

The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.

Isolation and sequencing of a gene coding for glyoxalase I activity from Salmonella typhimurium and comparison with other glyoxalase I sequences.

The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn2+ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.

Small molecule probes of glyoxalase I and glyoxalase II.

A number of synthetic tropolones and hydrophobic S-blocked glutathione analogues were investigated as potential inhibitors of glyoxalase I from Saccharomyces cerevisiae and glyoxalase II from bovine liver. Several tropolones containing a free C-2 hydroxy group were found to be potent inhibitors of glyoxalase I, whereas the glutathione conjugates were found to be modest to poor inhibitors of this enzyme. Most tropolones and glutathione conjugates, except 5-p-tolylazotropolone and S-carbobenzoxy-L-glutathione, were found to be poor inhibitors of glyoxalase II. A recent report on an extremely active glyoxalase system from Plasmodium falciparum suggested that several of the more potent inhibitors may have antimalarial properties. A number of these compounds in fact, exhibited antimalarial activity in the low micromolar range. Further studies are required to fully elucidate the mechanism(s) of the antimalarial properties of these compounds.

Investigation on glyoxalase I inhibitors.

Several classes of compounds - flavones, coumarins, S- and N-substituted glutathione analogs, transition state analogs, porphyrins, nucleotides and nucleosides - have been reported to inhibit the enzyme gloxalase I. In the current study, examination of some of the aforementioned compounds has revealed that squaric acid does not function as an inhibitor of glyoxalase I and several other compounds are much less effective in this regard than previously reported. Several new potent inhibitors of yeast glyoxalase I have been identified. Compounds containing the tropolone structure were especially inhibitory. Glutathione adducts of benzoquinone and naphthoquinone were also inhibitory and may be of particular interest with regard to the toxicology of normal aromatic metabolites in vivo.