Cloning and characterization of glyoxalase I from soybean.
Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No. X68819) resembling a GST being described as a glyoxalase I. To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures. When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or gamma-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean. When the clone X68819 was expressed in E. coli, 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 over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzymes 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%).
Skipsey
M
M
Department of Biological Sciences, University of Durham, Durham, DH1 3LE, UK.
Andrews
C J
CJ
Townson
J K
JK
Jepson
I
I
Edwards
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019281:Dimerization; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008322:Mammals; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D005982:Glutathione Transferase; D007791:Lactoylglutathione Lyase