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. 2004 Nov 15;384(Pt 1):111-7.
doi: 10.1042/BJ20041006.

Distinct classes of glyoxalase I: metal specificity of the Yersinia pestis, Pseudomonas aeruginosa and Neisseria meningitidis enzymes

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Distinct classes of glyoxalase I: metal specificity of the Yersinia pestis, Pseudomonas aeruginosa and Neisseria meningitidis enzymes

Nicole Sukdeo et al. Biochem J. .

Abstract

The metalloisomerase glyoxalase I (GlxI) catalyses the conversion of methylglyoxal-glutathione hemithioacetal and related derivatives into the corresponding thioesters. In contrast with the previously characterized GlxI enzymes of Homo sapiens, Pseudomonas putida and Saccharomyces cerevisiae, we recently determined that Escherichia coli GlxI surprisingly did not display Zn2+-activation, but instead exhibited maximal activity with Ni2+. To investigate whether non-Zn2+ activation defines a distinct, previously undocumented class of GlxI enzymes, or whether the E. coli GlxI is an exception to the previously established Zn2+-activated GlxI, we have cloned and characterized the bacterial GlxI from Yersinia pestis, Pseudomonas aeruginosa and Neisseria meningitidis. The metal-activation profiles for these additional GlxIs firmly establish the existence of a non-Zn2+-dependent grouping within the general category of GlxI enzymes. This second, established class of metal activation was formerly unidentified for this metalloenzyme. Amino acid sequence comparisons indicate a more extended peptide chain in the Zn2+-dependent forms of GlxI (H. sapiens, P. putida and S. cerevisiae), compared with the GlxI enzymes of E. coli, Y. pestis, P. aeruginosa and N. meningitidis. The longer sequence is due in part to the presence of additional regions situated fairly close to the metal ligands in the Zn2+-dependent forms of the lyase. With respect to sequence alignments, these inserts may potentially contribute to defining the metal specificity of GlxI at a structural level.

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Figures

Scheme 1
Scheme 1. Reactions of the glyoxalase system
Figure 1
Figure 1. Amino acid sequence alignment for H. sapiens, E. coli, Y. pestis, P. aeruginosa, N. meningitidis and P. putida GlxI enzymes
Conserved metal ligands are indicated in bold lettering with the position marked by an asterisk. The additional regions of the H. sapiens and P. putida GlxI enzymes are indicated in bold with underlining. The configuration of sequences in this alignment was obtained from a previous comparative study [29].
Figure 2
Figure 2. Electrospray ionization mass spectra of (A) Y. pestis (reconstructed spectrum) (B) P. aeruginosa GlxI and (C) N. meningitidis GlxI enzymes
The expected molecular masses of the monomeric enzymes are 14834 Da, 14251 Da and 15669 Da respectively.
Figure 3
Figure 3. Profiles of apoenzyme reactivation with various bivalent ions for (A) E. coli [19] (B) Y. pestis (C) P. aeruginosa and (D) N. meningitidis GlxI enzymes
To obtain the relative maximal activity values for these graphs, baseline apo-control assays were subtracted from all activities so that relative maximal activity was calculated using the specific activity−apo-specific activity values.
Figure 4
Figure 4. Titration of GlxI apoenzymes with various NiCl2 (black squares) and CoCl2 (grey circles) ratios: (A) E. coli [19], (B) Y. pestis, (C) P. aeruginosa and (D) N. meningitidis GlxI enzymes
Data points and error bars in (B), (C) and (D) represent the specific activity±S.D. for triplicate readings. Relative specific activity values were calculated as the proportion of activity relative to the highest specific activity values obtained during the NiCl2 titration.

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