Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis

Abstract

Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15's substrate preference. The X-ray crystal structure of the AtGH3.15·2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-catalyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.

Brassicaceae-specific Gretchen Hagen 3 acyl acid amido synthetases conjugate amino acids to chorismate, a precursor of aromatic amino acids and salicylic acid

“…This structure provides a view of how chorismate can bind in the acyl acid site of AtGH3.12/PBS3; however, the location of the ligand likely does not represent where it binds during catalysis, as the ring carboxylate of chorismate is ~10 Å away from the AMP. As noted for other GH3 protein structures (11)(12)(13), charge repulsion between the carboxylate of an acyl acid substrate and the nucleotide phosphate can alter positioning of ligands in the site. During the reaction sequence, the presence of Mg 2+ is an essential counterion that balances active site charges and allows the first half-reaction to occur (10,(30)(31).…”

“…5). Compared to the xray crystal structures of other GH3 acyl acid amido synthetase that use either auxins or jasmonic acid, key differences among the structures reveal how the AtGH3.12/PBS3 8 active site recognizes chorismate (10)(11)(12)(13)(28)(29). The active site of AtGH3.11/JAR1 has only one polar residue, a histidine, that interacts with the jasmonic acid ketone through a water molecule (10).…”

“…Although GH3 proteins are found in all plants, the roles of many of these proteins remains to be investigated. In Arabidopsis thaliana (thale cress), there are 19 GH3 proteins, and to date, only 8 of these have been biochemically characterized -AtGH3.1, AtGH3.2, AtGH3.3, AtGH3.5/WES1, AtGH3.11/JAR1, AtGH3.12/PBS3, AtGH3.15, and AtGH3.17/VAS2 (8)(9)(10)(11)(12)(13).…”

“…The second largest clade contains AtGH3.11/JAR1 and related proteins from multiple plants that function as jasmonyl-isoleucine synthetases to produce the bioactive jasmonate hormone (8,10). Recently, AtGH3.15 was found to modify the auxin precursor indole-3-butyric acid and auxin herbicides suggesting a role for GH3 proteins outside of the canonical plant hormones (12)(13).…”

“…tomato DC3000 infection assays, and a double mutant gh3.7/gh3.12 Arabidopsis line was generated to determine the combined role of this unique class of GH3 proteins in the Brassicaceae family. Due to the relaxed substrate specificity that is typical of GH3 proteins, it is not surprising that AtGH3.12/PBS3 can recognize both chorismate and isochorismate as substrates (11)(12)(13)24).…”