Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.
Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.
复制标题
利用黑腹果蝇玫瑰色突变株探究黄嘌呤脱氢酶的结构和功能。
DOI：
10.1042/bj2850507
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Robert C. Bray
中科院分区：
文献类型：
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作者：
Richard K. Hughes;W. Doyle;A. Chovnick;J. Whittle;Julian F. Burke;Robert C. Bray
研究了含钼羟基酶在黑腹果蝇玫瑰色突变株结构/功能研究中的作用。至少有23个这样的菌株可用，每个菌株对应于黄嘌呤脱氢酶序列中的一个已知氨基酸变化。通过序列比较，可以确定与该酶的铁硫中心和蝶呤钼辅因子相关的区域。通过使用各种氧化和还原底物，已经开发并严格测试了果蝇凝胶过滤提取物中不同的黄嘌呤脱氢酶催化活性的测定程序。这些方法已经应用于11个不同的玫瑰色突变株，这些突变株映射到序列的不同区域。所有研究的突变都会导致酶的特性活性变化。总的来说，这与公认的将辅因子分配到不同的区域以及钼、黄素和铁-硫中心的已知反应性是一致的。大多数结果都可以用只影响一个氧化还原中心的电子转移的突变来解释。活性数据提供了证据，证明FAD和NAD+/NADH结合位点在映射到黄素域的突变体中保留。因此，尽管序列比较有一些迹象，但我们得出结论，黄嘌呤脱氢酶这个结构域的结构不能与其他已有结构数据的黄素蛋白的结构直接相关。数据还表明，人工电子受体吩嗪甲硫酸盐作用于铁-硫中心，并表明这些中心可能不是钼和黄素之间电子转移所必需的。这项工作强调了对玫瑰色突变的黄嘌呤脱氢酶变异体进行遗传和生化联合研究在探索这类酶的结构和功能方面的重要性。
The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.