Overproduction and Characterization of a Dimeric Non-Zinc Glyoxalase I from Escherichia coli:  Evidence for Optimal Activation by Nickel Ions^,

Abstract

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+.

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

“…After extensive dialysis of the recombinant protein to remove loosely bound metals, 0.9 mol of nickel and 0.1 mol of zinc per mole of GLO1 dimer were detected by atomic absorption spectrophotometry. This stoichiometry suggests that one active site in the homodimeric protein does not tightly bind metal cofactor, an observation also made with the E. coli enzyme (7).…”

“…GLO1 is a metalloenzyme that isomerizes the glutathione hemithioacetal to S-D-lactoylglutathione through proton transfer to a metalbound enediol intermediate (5). This metal cofactor is zinc in eukaryotes but nickel in Escherichia coli (6,7). The difference in cofactor dependence is reflected in differences between the active sites of the human and E. coli enzymes, suggesting that the latter may be a target for antimicrobial therapy (8,9).…”

Glyoxalase biochemistry

“…This information provided structural confirmation of previous biophysical studies that indicated that Glo1 maintains an octahedral metal coordination environment, with metal-bound H 2 O/OH -also participating (55)(56)(57). Although also expecting the isolated Escherichia coli Glo1 to be a Zn 2+ -activated enzyme, a surprising metal activation profile was observed for this enzyme, drastically different from previously studied Glo1 (58). A narrower metal activation profile was observed for the E. coli Glo1, with Ni 2+ reconstitution providing the most active enzyme.…”

“…However, the coordination geometry was found to be close to trigonal bipyramidal, with only one H 2 O/OH -bound to the zinc center. Subsequent studies by nuclear magnetic resonance spectroscopy (NMR) elaborated on the inequivalence of the two active sites in the E. coli enzyme, accounting for previous solution studies (58,62,63). Additional studies on substrate thiol structure-function activities and kinetic isotope effects were reported for the E. coli Glo1 (64).…”

Allosteric Coupling of Two Different Functional Active Sites in Monomeric Plasmodium falciparum Glyoxalase I

“…1B). For example, GloI from human and yeast (4) and Plasmodium falciparum (5) prefers Zn 2ϩ , whereas GloI from several bacteria such as Escherichia coli (6) and Yersinia pestis, Pseudomonas aeruginosa, and Neisseria meningitidis (7) and from the protozoan parasite Leishmania major (8,9) is optimally activated in the presence of Ni 2ϩ . GloI from human (10,11) and E. coli (12) is active as a homodimer.…”