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 i nto their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyo xalase I (GlxI), catalyzes the isomerization of the hemithioacetal for med nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. T o study the Escherichia coli GlxI enzyme, the DNA encoding this protei n, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleo tide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and M-r of 14 919. The gloA gene has been overexpressed in E. co li and shown to encode for GlxI. An effective two-step purification pr otocol was developed, yielding 150-200 mg of homogeneous protein per l iter of culture. Electrospray mass spectrometry confirmed the monomeri c weight of the purified protein, while gel filtration analysis indica ted GU to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect o n 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 als o studied. This is the first example of a non-zinc activated GlxI whos e maximal activation is seen with Ni2+.