Dimerisation of maize glutathione transferases in recombinant bacteria.
Two cDNAs encoding novel type III maize (Zea mays) GST subunits, ZmGST VI and ZmGST VII, have been cloned in addition to the previously described ZmGST V. Together with the type I GSTs ZmGST I and ZmGST III, these subunits were expressed in Escherichia coli, both individually and in tandem combinations using a customised pET vector. The GST dimers formed were then characterised. When type I GSTs were co-expressed only the respective homodimers were formed rather than the ZmGST I-III heterodimer. The failure to form this heterodimer, together with the negligible herbicide-detoxifying activity associated with recombinant ZmGST III-III, suggests that the identity of herbicide-detoxifying isoenzymes described in maize as being composed of ZmGST III subunits requires re-evaluation. In contrast, co-expression of the type III GSTs ZmGST V and ZmGST VI resulted in the formation of ZmGST V-V, ZmGST VI-VI and ZmGST V-VI dimers in the ratio 1:1:2 as predicted for random subunit association. ZmGST V-VI had kinetic characteristics intermediate between those of the two homodimers, indicating that the subunits were catalytically independent of one another. Co-expression of ZmGST V and ZmGST VII resulted in the formation of ZmGST V-VII and this isoenzyme was subsequently identified in maize plants. Attempts to dimerise type I GST subunits with type III GST subunits proved unsuccessful. These results demonstrate the utility of co-expressing recombinant GSTs to explore the potential of subunit-subunit associations and to help unravel the complexity of homodimeric and heterodimeric GSTs in plants.
Dixon
D P
DP
Department of Biological Sciences, University of Durham, UK.
Cole
D J
DJ
Edwards
R
R
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase