Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase.
Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3'-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent Km for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster.
Matsson
M
M
Department of Microbiology, Lund University, Sölvegatan 12, S-22362 Lund, Sweden.
Ackrell
B A
BA
Cochran
B
B
Hederstedt
L
L
D002261:Carboxin / Q000494:pharmacology; D002952:Citric Acid Cycle / Q000502:physiology; D004352:Drug Resistance, Microbial; D042963:Electron Transport Complex II; D004591:Electrophoresis, Polyacrylamide Gel; D005659:Fungicides, Industrial / Q000494:pharmacology; D005798:Genes, Bacterial / Q000235:genetics; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics*; D010231:Paracoccus denitrificans / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics*; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology; D013997:Time Factors
D005659:Fungicides, Industrial; D009097:Multienzyme Complexes; D013804:Thenoyltrifluoroacetone; D002261:Carboxin; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase