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+.
Clugston
S L
SL
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Barnard
J F
JF
Kinach
R
R
Miedema
D
D
Ruman
R
R
Daub
E
E
Honek
J F
JF
D000595:Amino Acid Sequence; D001483:Base Sequence; D019281:Dimerization; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D007791:Lactoylglutathione Lyase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009532:Nickel; D012441:Saccharomyces cerevisiae
D009532:Nickel; D007791:Lactoylglutathione Lyase