ABSTRACT
Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides.
Subject(s)
Acetamides/metabolism , Acetamides/pharmacology , Cysteine/metabolism , Herbicides/metabolism , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Acetamides/chemistry , Acetyltransferases/metabolism , Acyltransferases/antagonists & inhibitors , Acyltransferases/genetics , Acyltransferases/metabolism , Amino Acid Sequence , Binding Sites , Carbon Radioisotopes , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Fatty Acid Elongases , Herbicides/chemistry , Inhibitory Concentration 50 , Isomerism , Malonyl Coenzyme A/analogs & derivatives , Malonyl Coenzyme A/metabolism , Molecular Sequence Data , Multienzyme Complexes/genetics , Peptide Fragments/analysis , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Plant Proteins/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
In the present study membrane fatty acids were analyzed to find a link between the biosynthesis inhibition of very-long-chain fatty acids and the phytotoxic effects of herbicidal chloroacetamides. Accordingly, we have isolated membranes of cucumber seedlings (Cucumis sativus) by two-phase partitioning and analyzed their fatty acid content. Saturated VLCFAs ranging from C20 to C26 were found in high amounts (22%) in the plasma membrane fraction. Non-modified VLCFAs were predominantly present in phospholipids, while saturated 2-hydroxylated VLCFAs were identified in cerebrosides. Treatment of intact seedlings with chloroacetamides markedly reduced the VLCFA content in the plasma membrane. This result could be specified by fatty-acid labeling using [14C]malonate as a substrate for fatty acid elongation. De novo incorporation of VLCFAs into the plasma membrane and into microsomal membranes, respectively, was severely impaired by chloroacetamides with I50 values between 10 to 100 nM. These results confirm the previous finding that chloroacetamides inhibit VLCFA biosynthesis localized in the microsomes (Böger et al., Pest Manage. Sci. 56, 497-508, 2000). The direct consequence of this inhibition is a strong decrease of VLCFAs required as constituents of the plasma membrane and the substitution by shorter acyl chains. Apparently, physical properties and function of the plasma membrane are affected eventually leading to death of the plant.
Subject(s)
Acetamides/pharmacology , Cell Membrane/drug effects , Membrane Lipids/metabolism , Cell Membrane/metabolism , Cucumis sativus , Fatty Acids/metabolism , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Phospholipids/metabolism , Plastids/metabolismABSTRACT
Indanofan and its analogs inhibited the elongation of stearoyl- or arachidoyl-CoA by [2-14C]-malonyl-CoA in leek microsomes from Allium porrum. Although the precise mode of interaction of indanofan at the molecular level is not completely clarified by the present study, it is concluded that indanofan and analogs act as inhibitor of the elongase enzyme involved in de novo biosynthesis of fatty acids with an alkyl chain longer than C18, called very-long-chain fatty acids (VLCFAs). For a strong inhibition of VLCFA formation chloro substituents at the benzene ring and the oxirane group were necessary. Furthermore, the greenhouse test showed strong activity for indanofan and its analogs, and the scores coincided with cell-free elongation inhibition. The cell-free assay, however, failed to indicate any activity for an analog having a methylene instead of the oxirane group, while both Digitaria ciliaris and Echinochloa oryzicola were killed with 1 kg a.i./ha. This finding cannot be discussed because the applied use rate of 1 kg a.i./ha is too high to allow for a score differentiation. For high concentrations of this compound additional unknown inhibitory effects may be involved besides fatty acid elongation.