Cloning and characterization of glyoxalase I from soybean

Glyoxalase I and glutathione transferase (GST) are two glutathione-dependen t enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between the se two enzymes has been suggested by a clone (Accession No. X68819) resembl ing a GST being described as a glyoxalase I. To characterize glyoxalase I i n soybean, GmGlyox I resembling the dimeric enzyme from animals has been cl oned from a cDNA library prepared from soybean suspension cultures. When ex pressed in Escherichia coil, GmGlyox I was found to be a 38-kDa dimer compo sed of 21-kDa subunits and unlike the enzyme from mammals showed activity i n the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathio ne, homoglutathione, or gamma-glutamylcysteine, showing no preference for h omoglutathione adducts over glutathione adducts, even though homoglutathion e is the dominant thiol in soybean. When the clone X68819 was expressed in E, coil, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3, GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione ov er homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzym es are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%). (C) 2000 Academic Press.