Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization

Jochen Kuper, Tracy Palmer, Ralf R Mendel, Gunter Schwarz

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    39 Citations (Scopus)

    Abstract

    The molybdenum cofactor (Moco). a highly conserved pterin compound coordinating molybdenum (Mo), is required for the enzymatic activities of molybdoenzymes. In all organisms studied so far Moco is synthesized by a unique and evolutionary old multistep pathway that requires the activities of at least six gene products. In eukaryotes, the last step of Moco synthesis, i.e., transfer and insertion of Mo into molybdopterin (MPT), is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals. Both domains (E and G) of these proteins are able to bind MPT in vitro. Here, we show the identification and mutational dissection of functionally important regions within the Cnx1 G domain that are essential for MPT binding, the conversion of MPT to Moco, and Moco stabilization. By functional screening for mutants in the Cnx1 G domain that are no longer able to complement Escherichia coil mogA mutants, we found two classes of mutations in highly conserved amino acid residues. (i) The first class affects in vitro binding of MPT to the protein and the stabilization of Moco, the product of the G domain. (ii) The second class is represented by two independent mutations in the aspartate 515 position that is not affected in MPI binding and Moco stabilization; rather the conversion of MPT to Moco by using bound MPT and a yet unknown form of Mo is completely abolished. The results presented here provide biochemical evidence for a purified Cnx1 G domain catalyzing the insertion of Mo into MPT.

    Original languageEnglish
    Pages (from-to)6475-6480
    Number of pages6
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume97
    Issue number12
    DOIs
    Publication statusPublished - 6 Jun 2000

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