Rapid and Reversible High-Affinity Binding of the Dinitroaniline Herbicide Oryzalin to Tubulin from Zea mays L

Hugdahl, Jeffrey D.; Morejohn, Louis C.

doi:10.1104/pp.102.3.725

Cited by 140 publications

(129 citation statements)

References 32 publications

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“…For each ␣-tubulin conformation, we found the consensus binding site ( Figure 5A) was both the largest cluster and contained the binding conformations with the highest affinity. The binding affinity predicted for this site was 23 nM and is in agreement with experimentally determined values of 95-117 nM obtained with purified plant tubulin (Hugdahl and Morejohn, 1993;Murthy et al, 1994). The site identified by our docking simulations is located beneath the N loop and is composed of residues Arg2, Glu3, Val4, Trp21, Phe24, His28, Ile42, Asp47, Arg64, Cys65, Thr239, Arg243, and Phe244 ( Figure 5A).…”

supporting

confidence: 71%

“…Dinitroanilines also disrupt the microtubules of protozoa, including both free-living species such as Tetrahymena and protozoan parasites such as Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii (Chan and Fong, 1990;Chan et al, 1991;Gu et al, 1995;Edlind et al, 1996;Stokkermans et al, 1996;Armson et al, 1999; Makioka et al, 2000a,b;Traub-Cseko et al, 2001). Remarkably, the activity of dinitroanilines is restricted to plants and protozoa; these compounds are ineffective against vertebrate or fungal microtubules (Chan and Fong, 1990;Hugdahl and Morejohn, 1993;Murthy et al, 1994;Edlind et al, 1996).T. gondii is a member of the Apicomplexa, a phylum of parasites that includes several medically and agriculturally significant pathogens (Black and Boothroyd, 2000).…”

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confidence: 99%

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Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

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Sept

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Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in ␣-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of ␣-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma ␣-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus ␣-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate ␣-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on ␣-tubulin function. INTRODUCTIONMicrotubules are polymers constructed from ␣-␤-tubulin heterodimers (Downing and Nogales, 1998a,b). These structures are rapidly assembled and disassembled to create essential components of eukaryotic cells, such as spindles and flagella. The dynamic nature of microtubules makes them susceptible to pharmacological agents. Microtubule-disrupting and microtubule-stabilizing drugs have provided great insight into tubulin and microtubule function; they also have tremendous practical use. Compounds that perturb microtubule dynamics are currently some of the most effective drugs to treat medical conditions, including cancer, gout, and helminth infection (Jordan et al., 1998). Dinitroanilines (oryzalin, ethafluralin, and trifluralin) disrupt the microtubules of plants, ranging from the single-celled alga Chlamydomonas reinhardtii to higher plants such as the monocot Eleusine indica (James et al., 1993;Anthony et al., 1998;Zeng and Baird, 1999). Dinitroanilines also disrupt the microtubules of protozoa, including both free-living species such as Tetrahymena and protozoan parasites such as Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii (Chan and Fong, 1990;Chan et al., 1991;Gu et al., 1995;Edlind et al., 1996;Stokkermans et al., 1996;Armson et al., 1999; Makioka et al., 2000a,b;Traub-Cseko et al., 2001). Remarkably, the activity of dinitroanilines is restricted to plants and protozoa; these compounds are ineffective against vertebrate or fungal microtubules (Chan and Fong, 1990;Hugdahl and Morejohn, 1993;Murthy et al., 1994;Edlind et al., 1996).T. gondii is a member of the Apicomplexa, a phylum of parasites that includes several medically and agriculturally significant pathogens (Black and Boothroyd, 2000). Apicomplexans are obligate intracell...

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supporting

confidence: 71%

mentioning

confidence: 99%

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Morrissette

Mitra

Sept

et al. 2004

MBoC

135

153

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“…To inhibit microtubule function partially, we chose oryzalin, because its interaction with plant tubulin has been well characterized (Hugdahl and Morejohn, 1993). Nevertheless, with any inhibitor, there may be nonspecific effects; therefore, we compared oryzalin to microtubule inhibitors with other chemical structures (Vaughn and Lehnen, 1991).…”

Section: Determination Of Suitable Inhibitor Concentrationsmentioning

confidence: 99%

“…In contrast to inhibiting microtubules totally, which gives rise to more or less isotropic expansion, we hypothesized that inhibiting them partially would stimulate tangential expansion modestly, making it easier to measure, but not enough to make expansion isotropic. The biochemistry of interaction between the microtubule inhibitor, oryzalin, and plant tubulin has been well characterized (Hugdahl and Morejohn, 1993) and partial inhibition at low concentration minimizes the possibility of nonspecific effects. We used low concentrations of oryzalin, which allowed growth to continue at a reduced but virtually steady rate for several days, and quantified profiles of longitudinal and tangential expansion rate and, concomitantly, the spatial profile of microfibril alignment.…”

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Disorganization of Cortical Microtubules Stimulates Tangential Expansion and Reduces the Uniformity of Cellulose Microfibril Alignment among Cells in the Root of Arabidopsis

et al. 2004

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To test the role of cortical microtubules in aligning cellulose microfibrils and controlling anisotropic expansion, we exposed Arabidopsis thaliana roots to moderate levels of the microtubule inhibitor, oryzalin. After 2 d of treatment, roots grow at approximately steady state. At that time, the spatial profiles of relative expansion rate in length and diameter were quantified, and roots were cryofixed, freeze-substituted, embedded in plastic, and sectioned. The angular distribution of microtubules as a function of distance from the tip was quantified from antitubulin immunofluorescence images. In alternate sections, the overall amount of alignment among microfibrils and their mean orientation as a function of position was quantified with polarized-light microscopy. The spatial profiles of relative expansion show that the drug affects relative elongation and tangential expansion rates independently. The microtubule distributions averaged to transverse in the growth zone for all treatments, but on oryzalin the distributions became broad, indicating poorly organized arrays. At a subcellular scale, cellulose microfibrils in oryzalin-treated roots were as well aligned as in controls; however, the mean alignment direction, while consistently transverse in the controls, was increasingly variable with oryzalin concentration, meaning that microfibril orientation in one location tended to differ from that of a neighboring location. This conclusion was confirmed by direct observations of microfibrils with field-emission scanning electron microscopy. Taken together, these results suggest that cortical microtubules ensure microfibrils are aligned consistently across the organ, thereby endowing the organ with a uniform mechanical structure.How do plants build organs with specific and heritable shapes? A part of the answer to this question lies in the control of growth. It is not growth rate per se that is crucial for morphogenesis but the directionality of growth. If growth rate were the same in all directions, i.e. isotropic, plant organs would be spherical; organs attain shapes other than spherical because their component cells grow at different rates in different directions, i.e. anisotropically. Understanding how cells control the anisotropy of their expansion is essential for understanding morphogenesis.Expansion anisotropy is characterized by the direction in which the maximal growth rate occurs and by the degree to which the maximum differs from the minimum. The direction of maximal expansion rate is known to be controlled by the direction of net alignment of cellulose microfibrils. Within a growing cell wall, microfibrils are aligned, on average, perpendicularly to the direction of maximal expansion rate, and the aligned cellulose microfibrils confer a mechanical anisotropy on the cell wall, which translates into expansion anisotropy (Green, 1980;Taiz, 1984). The wall can be considered a composite material with strong, rod-shaped particles (microfibrils) embedded in a compliant matrix. Such materials deform perpendic...

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“…Microtubules are a known target of fungicides such as the benzimidazoles. Since some fungicides and herbicides display unacceptable phytotoxicity, 3,4) it is important that any antimitotic compound for plant pathogenic fungi should be non-phytotoxic.…”

mentioning

confidence: 99%