Matsson M, Ackrell B A, Cochran B, Hederstedt L
Department of Microbiology, Lund University, Sölvegatan 12, S-22362 Lund, Sweden.
Arch Microbiol. 1998 Jul;170(1):27-37. doi: 10.1007/s002030050611.
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.
醌还原酶是柠檬酸循环和呼吸链中的一种膜结合酶。萎锈灵是某些生物体中该酶的有效抑制剂。已发现反硝化副球菌在体内对萎锈灵敏感，并分离出了在3'-甲基萎锈灵存在下生长的突变体。这些突变体的膜显示出对萎锈灵有抗性的琥珀酸：醌还原酶活性。在四个突变体中鉴定出了赋予萎锈灵抗性的突变。它们在sdhD基因中含有相同的错义突变，该基因编码琥珀酸：醌还原酶复合物的两个膜内在多肽之一。该突变导致SdhD多肽第89位的天冬氨酸被甘氨酸取代。构建了过量表达野生型或突变型酶的反硝化副球菌菌株。分析了纯化酶的酶学性质。对醌（DPB）的表观Km和对噻吩甲酰三氟丙酮的敏感性对于抗萎锈灵的酶来说是正常的，但琥珀酸：醌还原酶活性低于野生型酶。赋予萎锈灵抗性的突变表明了抑制剂在酶上结合的区域。先前报道的玉米黑粉菌琥珀酸：醌还原酶的铁硫蛋白中靠近[3Fe-4S]簇的组氨酸被亮氨酸取代，赋予了对萎锈灵和噻吩甲酰三氟丙酮的抗性。在本研究中鉴定出的反硝化副球菌SdhD多肽中天冬氨酸被甘氨酸取代，赋予了对萎锈灵但不包括对噻吩甲酰三氟丙酮的抗性，该取代位于预测的连接两个跨膜区段的胞质环中。这个环很可能位于[3Fe-4S]簇附近。
