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Löffler, M., Knecht, W., Rawls, J., Ullrich, A., Dietz, C. (2002). Drosophila melanogaster dihydroorotate dehydrogenase: the N-terminus is important for biological function in vivo but not for catalytic properties in vitro.  Insect Biochem. Mol. Biol. 32(9): 1159--1169.
FlyBase ID
FBrf0152076
Publication Type
Research paper
Abstract

Dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis, is an integral flavoprotein of the inner mitchondrial membrane and is functionally connected to the respiratory chain. Here, experiments have been directed toward determining the roles of the N-terminal sequence motifs both in enzymatic properties of insect DHODH produced in vitro and the in vivo function of the protein. Full-length and three N-terminal truncated derivatives of the Drosophila melanogaster enzyme were expressed in Escherichia coli and purified. For identification on Western blots of recombinant DHODH as well as the native enzyme from flies polyclonal anti-DHODH immunoglobulins were generated and affinity-purified. The enzymatic characteristics of the four versions of DHODH were very similar, indicating that the N-terminus of the enzyme does not influence its catalytic function or its susceptibility to prominent DHODH inhibitors: A77-1726, brequinar, dichloroallyl-lawsone and redoxal. Whereas the efficacy of A77-1726 and dichloroallyl-lawsone were similar with Drosophila and human DHODH, that of brequinar and redoxal differed significantly. The differences in responses of insect DHODH and the enzyme from other species may allow the design of new agents that will selectively control insect growth, due to pyrimidine nucleotide limitation. In vivo expression of the full-length and N-truncated DHODHs from engineered transgenes revealed that the truncated proteins could not support normal de novo pyrimidine biosynthesis during development of the fly (i.e., failure to complement dhod-null mutations), apparently due to instability of the truncated proteins. It is concluded that the proper intracellular localization, directed by the N-terminal targeting and transmembrane motifs, is required for stability and subsequent proper biological function in vivo.

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    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Insect Biochem. Mol. Biol.
    Title
    Insect Biochemistry and Molecular Biology
    Publication Year
    1992-
    ISBN/ISSN
    0965-1748
    Data From Reference
    Aberrations (1)
    Alleles (8)
    Genes (1)
    Transgenic Constructs (6)