Isolation of mutants of the Arabidopsis thaliana alternative oxidase (ubiquinol:oxygen oxidoreductase) resistant to salicylhydroxamic acid.
The plant-type ubiquinol:oxygen oxidoreductase, commonly called the alternative oxidase, is a respiratory enzyme thought to contain non-heme iron at its active site. To explore the structure of the enzyme by identifying amino acids involved in inhibitor-binding, a library of random mutants of the Arabidopsis thaliana alternative oxidase was constructed using error-prone polymerase chain reaction and expressed in the heme-deficient Escherichia coli SASX41B. Selection for resistance to salicylhydroxamic acid (SHAM) resulted in the recovery of four mutations. Three of these, F215L, M219I, and M219V, confer a small, but measurable resistance to SHAM of between 1.4- and 1.7-fold relative to the wild type alternative oxidase. These changes are located in a putative amphipathic helix following the second transmembrane helix. The fourth mutation, G303E, is found three residues from the C-terminus of the protein, and results in 4. 6-fold resistance to SHAM. None of the mutations have any effect on the sensitivity of the alternative oxidase to propyl gallate. The identification of distant residues involved in SHAM resistance suggests that the poorly conserved C-terminal region is in spatial proximity to the amphipathic helix, and thus located in the vicinity of the iron-binding motif.
Berthold
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Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA. berthold@uiuc.edu
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010940:Plant Proteins; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012457:Salicylamides / Q000494:pharmacology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014345:Trypanosoma; D000099188:Alternative Oxidase
D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D012457:Salicylamides; D000099188:Alternative Oxidase; C005703:salicylhydroxamic acid