An enzyme-directed approach has led to finding specific inhibitors of imidazoleglycerolphosphate dehydratase (IGPD), which catalyzes a dehydration reaction of imidazoleglycerol phosphate (IGP) to form imidazoleacetol phosphate in the biosynthesis of histidine. These newly synthesized IGPD inhibitors showed wide-spectrum, postemergence herbicidal activity at application rates ranging from 0.05 to 2 kg ai ha−1. IGPD from wheat germ was purified to apparent homogeneity and characterized, and IGPD cDNAs were isolated from Arabidopsis and wheat cDNA libraries. Both the purified plant enzyme and the recombinant enzymes, which have been prepared by expressing the isolated IGPD cDNAs in the baculovirus/insect cell system, were used for establishment of an in vitro enzyme assay for inhibitor evaluation. Enzyme inhibitors were designed and synthesized by considering the structure of the substrate, IGP, and the inhibition activities were enhanced by a systematic structure—activity relationship study in vitro. Three triazole phosphonates, IRL 1695, IRL 1803, and IRL 1856, inhibited the reaction of the enzyme with Ki
values of 40 ± 6.5 nM, 10 ± 1.6 nM, and 8.5 ± 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. Plant cell growth inhibition was completely reversed only by l-histidine, proving that the cytotoxicity was primarily caused by the inhibition of histidine biosynthesis. These new IGPD inhibitors have demonstrated that the specific inhibition of plant IGPD is lethal to plants. Further, treatment of Arabidopsis with IRL 1803 affected gene expression of eight enzymes involved in the biosynthesis of aromatic amino acids, histidine, lysine, and purines. These effects were reversed by the addition of l-histidine, indicating that the changes in gene expression were caused by the inhibition of l-histidine biosynthesis. These results indicate that higher plants, like microorganisms, are capable of cross-pathway metabolic regulation.