Forced Evolution of a Herbicide Detoxifying Glutathione Transferase

Abstract

Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107-119, which contribute to herbicide binding.The relative rate of herbicide detoxification in crops and weeds is a primary determinant of their selectivity (1). Crops rapidly metabolize herbicides by oxidative or hydrolytic reactions followed by conjugation with sugars or peptides and vacuolar sequestration of the polar products (1). In weeds, these detoxification reactions are slower. Glutathione S-transferases (GSTs) 1 have a well characterized role in determining the metabolism and selectivity of chloroacetanilide, thiocarbamate, and chloro-s-triazine herbicides in maize (2). GSTs catalyze the conjugation of these herbicides with the tripeptide glutathione (␥-glutamyl-cysteinyl-glycine) to form non-toxic S-glutathionylated products (2). In other crops, alternative GST activities determine herbicide selectivity. For example, photobleaching diphenylether herbicides such as fluorodifen are rapidly detoxified by GSTs in legumes (see Fig. 1A) but less efficiently in maize (3). Species-dependent GST-mediated detoxification can be explained by differences in the expression of the six distinct families of plant GST genes, classified as the Phi, Zeta, Tau, Theta, Lambda, and dehydroascorbate reductase classes (4 -6). The plant-specific Phi and Tau GSTs are primarily responsible for herbicide detoxification, showing class specificity in substrate preference. Phi enzymes (GSTFs) are highly active toward chloroacetanilide and thiocarbamate herbicides, whereas the Tau enzymes (GSTUs) are efficient in detoxifying diphenylethers and aryloxyphenoxypropionates (7,8). In maize, GSTFs are the major class of expressed GST (7), whereas in soybean, GSTUs predominate, with this difference accounting for the differential detoxification of different classes of herbicide in the two crops (4).We have been interested in enhancing the detoxifying potential of pl...

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

“…GST Expression in Plants-Transgenic Arabidopsis lines constitutively overexpressing cytosolic ZmGSTU1 and ZmGSTU2 were available from previous studies (22). For transient cytosolic expression, ZmGSTU1 and ZmGSTU2 were cloned into a custom binary vector allowing constitutive expression of N-ter-minal Strep-tagged (BIN-STRP3) protein (see the supplemental material).…”

“…Modeling-Modeling of PPgen binding to ZmGSTU1 was carried out using the protein coordinates of OsGSTU1, PDB code 1OYJ (22). The OsGSTU1 polypeptide is 73% sequenceidentical to ZmGSTU1 and highly conserved in sequence around the active site cavity and was used directly for modeling.…”

“…1. The 680-bp NcoI-Hind III EFD6-115A and 770-bp NdeIHindIII AtGSTT1 fragments corresponding from plasmids pEFD6-115A (encoding a mutant ZmGSTU) and pET-ATT4-2 (AtGSTT1), respectively (22,25), were cloned into plastid expression cassettes driven by the AtpA promoter and terminated by the rbcL 3Ј-untranslated region (26) (supplemental Fig. 1).…”

Creation of herbicide-tolerant crops by gene targeting

“…Structure analysis (crystal analysis), molecular modeling and mutagenesis studies provide an understanding of the molecular basis of GST-catalyzed herbicide binding and how single amino acid substitution(s) can improve GST catalytic efficiency and affect substrate specificity for herbicides and xenobiotics. [79][80][81] GOX, GAT, DMO, and ADD genes used for creating herbicide-tolerant crops also encode non-target site herbicide-tolerant proteins, but these transgenes are derived from microorganisms. When endogenous counterparts of these genes in crops are revealed and the herbicide metabolic mechanism is clarified, GT can be applied to create non-target site herbicide-tolerant crops by modifying these endogenous genes.…”

The Outlook for Protein Engineering in Crop Improvement

“…Dixon et al (2003) applied a forced evolution strategy to two maize glutathione-S-transferases and isolated seven mutants with enhanced fluorodifen detoxifying activity, the best of which contained a single point mutation and had 19-fold higher activity than the parent enzymes. Further targeted mutagenesis of the point mutant yielded an enzyme that had 29-fold higher activity.…”