Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase (complex II) |
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Authors: | Mikael Matsson Brian A C Ackrell Bruce Cochran L Hederstedt |
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Institution: | Department of Microbiology, Lund University, S?lvegatan 12, S-22362 Lund, Sweden e-mail: lars.hederstedt@mikrbiol.lu.se Tel. +46-46-2228622; Fax +46-46-157839, SE Molecular Biology Division, Veterans Administration Medical Center, University of California, San Francisco, CA 94121, USA, US
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Abstract: | 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 K
m 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.
Received: 18 November 1997 / Accepted: 13 February 1998 |
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Keywords: | Succinate:quinone reductase Succinate dehydrogenase Paracoccus denitrificans Quinone Carboxin Thenoyltrifluoroacetone Electron transport |
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