Herbicidal Activity of an Isopropylmalate Dehydrogenase Inhibitor

Abstract

With the discovery of ALS as the site of action of the sulfonylurea and imidazolinone herbicides (LaRossa and Schloss, 1984;Ray, 1984;Shaner et al., 1984), considerable attention tumed to other enzymes in branched-chain amino acid biosynthesis as potential herbicidal target sites (Schulz et al., 1988; Pirmng, 1989;Aulabaugh and Schloss, 1990;Schloss and Aulabaugh, 1990;Wittenbach et al., 1991 Wittenbach et al., , 1992Hawkes, 1993). In attempting to improve the herbicidal activity of some KARI inhibitors (Wittenbach et al., 1991), we discovered a class of chemicals with increased herbicidal activity that had essentially no in vitro activity on the plant enzyme. In follow-up studies using a pea root growth assay (Ray, 1984), we found that Leu alone could prevent the inhibitory effect of the new compounds (Wittenbach et al., 1992), suggesting that they inhibited a step in the Leu pathway. When the intermediates of Leu biosynthesis were assayed for their ability to prevent the inhibition, it was found that only Leu and 2-ketoisocaproate were able to protect the roots from inhibition. This suggested that IPMDH was the site of action. The sensitivity of this enzyme to O-IbOHA was then confirmed using purified IPMDH from Salmonella typhimurium (Wittenbach et al., 1992). This paper describes some of the characteristics of the plant enzyme and the proposed mechanism of inhibition for 0-IbOHA. In addition, we have compared enzyme inhibition with herbicidal activity for severa1 plant species. Based on these results, inhibition of IPMDH is confirmed as the site of action for the herbicide O-IbOHA. Possible explanations for the herbicidal activity and selectivity of this compound are presented. MATERIALS AND METHODS Enzyme lsolationIPMDH was extracted from light-grown (growth chamber set at 22OC, 16-h day at a PPFD of 200 pmol m-* s-' ; 16OC, 8-h night) shoots of pea (Pisum sativum L. cv Early Frosty), soybean (Glycine max L. Merr. cv Williams), and moming glory (Ipomoea purpurea [L.] Roth) and from etiolated (22OC dark room) shoots of com (Zea mays L. cv Funks G4689), giant foxtail (Setaria faberi), and pea (the enzyme from etiolated tissue had the same properties as that from light-grown tissue and the extract was spectrophotometrically cleaner). Seedlings were grown in Metro-Mix 350 (Grace/Sierra Horticultural Products Company, Milpitas, CA) and the shoots were harvested when they were 8 to 16 cm tall. (IPMDH was nearly equally distributed throughout the shoot and root of these seedlings, and there appeared to be no difference between the enzyme of the shoot and that of the root.) Plant shoots were extracted at 4OC in 0.1 M potassium phosphate buffer (1:2, w/v), pH 7.0, containing 1 m DTT and 1 m MgC12 using a Waring blender. The homogenate was filtered through three layers of cheesecloth, and solid (NH4)*S04 was added with stirring to give 30% saturation. The extracts were then centrifuged at 12,OOOg for 30 min, and the supematant was saved. Ammonium sulfate was then added to the supernatant to yield 65% saturation, an...

Evidence for cross-pathway regulation of metabolic gene expression in plants.

“…Over a 60-hr time course, during which the tissue became chlorotic and failed to grow, none of the genes examined in Fig. 3 (33).…”

Putative Nitrogen Sensing Systems in Higher Plants

“…Consistent with this observation, A. thaliana mutants defective in BBMII (N '-[(5'-phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) isomerase in the histidine biosynthetic pathway also had a 2-fold upregulation in total free amino acid content at the juvenile stage (Noutoshi et al 2005). Similarly, blocking leucine biosynthesis through specific inhibition of isopropylmalate dehydrogenase also results in the accumulation of other branched chain amino acids, as well as the aromatic amino acids tyrosine and phenylalanine (Wittenbach et al 1994). Indeed, many reports have indicated that aromatic amino acid biosynthetic genes are subject to cross-pathway regulation.…”

The High-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis

“…Pisum sativum L.) with apparent inhibitor constants in the nanomolar range. 21 The small molecule docking program GOLD was used to investigate the binding mode of O-ibOHA. 22 As a first test for the reliability of the docking results, the substrate IPM was docked into the active site of Mtb-IPMDH and the proposed binding mode was compared with the experimentally observed IPM position in the T. ferrooxidans-IPMDH crystal structure.…”