Biochemical and Structural Characterization of Salmonella typhimurium Glyoxalase II:  New Insights into Metal Ion Selectivity^,

Abstract

Glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-beta-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 A shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II.

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

“…This ability for catalysis to proceed with similar efficiency in the presence of different metal ions has also been observed in other bacterial GLX2 enzymes. 11,20 It may also indicate that the extensive hydrogen bonding network of liganding residues is able to undergo considerable flexibility in accommodating different metal ions, which is perhaps reflective of the requirement on the part of the organism to rapidly adapt to different environmental conditions.…”

“…Both histidine ligands form contacts through the Ne2 atom, in full agreement with the His-binding mode observed among the metallo-b-lactamase superfamily members. 11,17,20 One common structural feature of enzymes that have a metallo-b-lactamase fold is an extensive Hbonding network around the metal-binding ligands in the form of a so-called ''second shell'' of interactions. As YcbL represents the first example of a GLX2 enzyme with a single metal center, it is informative to compare the geometries of the secondshell amino acid metal ligands.…”

“…20 The hydroxyl of the Y145 in the human enzyme interacts with a glutathione carbonyl, and, interestingly, this interaction is absent in the plant and bacterial GLX2 structures that have been determined to date.…”

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

“…The metal ions are thought to simultaneously polarize the PϭO or CϭO bonds of their respective substrates and lower the pK a of the attacking water molecule, which is typically sandwiched between the two metals. A range of metal ions are utilized in the ␤-lactamase family, most commonly Zn 2ϩ , Fe 3ϩ , and Mn 2ϩ , with some individual enzymes displaying activity with all three of these metal ions (30), and mixed metal pairs (31). PhnP is notable in that it has a distinct preference for Ni 2ϩ and Mn 2ϩ ions for hydrolysis of bpNPP, whereas Zn 2ϩ affords considerably lower activity.…”

Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg257/Lys260, and unmasking of acid-base catalysis

“…protein is a member of the structurally diverse group of binuclear metallohydrolases (Cameron et al, 1999;Marasinghe et al, 2005;Campos-Bermudez et al, 2007). The active site can be separated into the catalytic center where hydrolysis takes place and the substrate-binding site.…”