Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases.
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.
Eckermann
Christian
C
Institut für Biologie II, Universität Freiburg, D-79104 Freiburg, Germany.
Matthes
Bernd
B
Nimtz
Manfred
M
Reiser
Verena
V
Lederer
Barbara
B
Böger
Peter
P
Schröder
Joachim
J
D000081:Acetamides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D000123:Acetyltransferases / Q000378:metabolism; D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003545:Cysteine / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000081031:Fatty Acid Elongases; D006540:Herbicides / Q000737:chemistry / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007536:Isomerism; D008316:Malonyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D000081:Acetamides; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D006540:Herbicides; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; C013874:chloroacetamide; D008316:Malonyl Coenzyme A; D000217:Acyltransferases; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases; C121200:p-coumaroyltriacetic acid synthase; C022674:stilbene synthase; C020141:flavanone synthetase; D003545:Cysteine