Untitled

Abstract

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.

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

“…Protoporphyrinogen IX (PPgen) was freshly synthesized from protoporphyrin IX (PPIX) by reduction with sodium amalgam after dissolution in 20% ethanol containing 10 mM KOH (19). Constructs pDD5 and GST6, for expressing N-terminal ␤-galactosidase fusions of ZmGSTU1 and ZmGSTU2, respectively, in E. coli, were available from previous studies (15,20), as were chimeras encoding a series of spliced variants of the two ZmGSTUs (21) and pET11-based vectors for expression of untagged maize GSTs (20). Vectors for expressing ZmGSTU1 and ZmGSTU2 as N-terminal His-tagged polypeptides (ZmGSTU1-his and ZmGSTU2-his, respectively) were synthesized by ligating the coding sequences for the enzymes into the NdeI and BamH1 sites of pET-28a (Novagen).…”

Forced Evolution of a Herbicide Detoxifying Glutathione Transferase

“…Ϫ vectors containing cDNA sequences for ZmGSTU1 and ZmGSTU2, respectively, were obtained from cDNA expression library antibody screens as detailed previously (9,10). In the work describing their original isolation, ZmGSTU1 was termed ZmGSTV and ZmGSTU2 was termed ZmGSTVI, the new designations reflecting a unifying change in nomenclature (5).…”

“…Analysis of Recombinant Mutant GSTs-GSTUs cloned in either pBluescript or pET plasmids in Escherichia coli were cultured without isopropyl-1-thio-␤-D-galactopyranoside induction, and the respective recombinant proteins were purified using S-hexylglutathione-Sepharose (9,10). Purified GSTs were extensively dialyzed to remove S-hexylglutathione and assayed for activity toward CDNB (1-chloro-2,4-dinitrobenzene) and fluorodifen (12), the latter by following the increase in absorbance at 400 nm after incubation at 30°C in 0.1 M glycine-NaOH buffer, pH 9.5, containing 5 mM glutathione and 50 M fluorodifen.…”

“…In maize (Zea mays L.), ZmGSTU1 and ZmGSTU2 are the major Tau enzymes expressed, although considerably less abundant than the ZmGSTFs (9,10). With the intention of enhancing their detoxifying activity toward fluorodifen, we have used reconstructive error-prone PCR (11) to randomly generate mutant GSTUs derived from both ZmGSTU1 and ZmGSTU2.…”

A Plant-specific Tau Class Glutathione S-transferase from Oryza sativa Having Significant Detoxification Activity Towards Chloroacetanilide Herbicides

“…12,13 In addition, the OsGSTU3 showed low activities towards the diphenyl ether herbicides, acifluorofen and fluorodifen. However, the tau class GSTs in maize and soybean had significant activity towards photobleaching herbicides, such as diphenyl ethers.…”