Structural and biochemical characterization of glyoxalase II from Salmonella typhimurium LT2

CAMPOS BERMUDEZ, VALERIA A. ; LEITE, NEY RIBEIRO ; KROG, RENATA; COSTA FILHO, ANTONIO J.; OLIVA, GLAUCIUS ; SONCINI, FERNANDO C. , AND VILA, ALEJANDRO J.

Rosario, Santa Fe, Argentina.

Congreso; XXXV Annual Meeting of the Argentinean Biophysical Society; 2006

The glyoxalase system consists of two enzymes, lactoylglutathione lyase (glyoxalase I, Glx I) and hydroxyacylglutathione hydrolase (glyoxalase lI, Glx II) that act coordinately to convert a variety of a-keto aldehydes into hydroxyacids in the presence of glutathione. GLX II is part of the metallo-b-lactamase (MBL) superfamily. The members of this superfamily are characterized by the presence of an ab/ba fold and a conserved motif able to bind up to two metal ions in their active sites [1]. Variations on the sequence elements of this motif have been correlated with the selectivity towards different metal ions, for instance Arabidopsis cytoplasmic GLX II can bind zinc, iron, and manganese, its mitochondrial isoform is a iron/zinc protein, while the human enzyme was reported to bind two zinc ions [2]. We have identified in the Salmonella typhimurium LT2 genome an ORF showing a significant homology sequence with glyoxalases II, containing the putative metal binding motif and protein fold of MBL superfamily [3]. This ORF was PCR-amplified from chromosomal DNA and cloned in a pET32 expression vector to generate a fusion protein to thioredoxin (Trx-tag) and His-Tag sequence. The resulting protein was overexpressed and purified using a Ni-agarose resin. We named this protein GloB. GloB was able to hydrolyze S-D-lactoylglutathione, confirming its predicted identity as glyoxalase II. This activity was dependent on the presence of divalent metal ion and it was established by the absence of hydrolysis when EDTA was added to the reaction medium.To identify the native metal of GloB, the protein was overexpressed in a rich medium and in minimal medium supplemented with different metals.  We found that this protein is able to bind iron, zinc as well as manganese in its bimetallic center, as revealed by atomic absorption measurements, 1H-NMR and EPR.X-ray crystallography of GloB showed that the protein presents the predicted ab/ba fold with the putative metal binding ligands involved in the coordination of the metal ions in a bimetallic center.