USE OF ROSY MUTANT STRAINS OF DROSOPHILA-MELANOGASTER TO PROBE THE STRUCTURE AND FUNCTION OF XANTHINE DEHYDROGENASE

被引:29
作者
HUGHES, RK [1 ]
DOYLE, WA [1 ]
CHOVNICK, A [1 ]
WHITTLE, JRS [1 ]
BURKE, JF [1 ]
BRAY, RC [1 ]
机构
[1] UNIV CONNECTICUT, MOLEC & CELL BIOL LAB, STORRS, CT 06269 USA
关键词
D O I
10.1042/bj2850507
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 oxidizin 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 molybden 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.
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页码:507 / 513
页数:7
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