Arabidopsis thaliana fatty acid alpha-dioxygenase-1: evaluation of substrates, inhibitors and amino-terminal function

W. Liu, L. H. Wang, P. Fabian, Y. Hayashi, C. M. McGinley, W. A. van der Donk, R. J. Kulmacz

Research output: Contribution to journalArticlepeer-review


Plant alpha dioxygenases (PADOX) convert fatty acids to 2-hydroperoxy products that are important in plant signaling pathways. The PADOX amino-terminal domain is distinct from that in other myeloperoxidase-family hemoproteins, and the positional specificity and prosthetic group of PADOX distinguish them from the non-heme iron plant lipoxygenases. The constraints of the PADOX active site on potential substrates are poorly understood and only limited structure-function and mechanistic information is available for these enzymes. We developed several bacterial and insect cell systems for expression of recombinant Arabidopsis thaliana PADOX1 and evaluated the enzyme's substrate and inhibitor profiles and explored the functional role of the amino-terminal domain. Substrate specificity studies gave the following relative oxygenase activity values: linolenate, 1.00; linoleate, 0.95; oleate, 0.84; palmitoleate, 0.69; myristate, 0.23; palmitate, 0.17; and gamma-linolenate, 0.16. Methyl esters of myristate, linoleate and linolenate were not oxygenated. 3-Thiamyristate was the only oxygenase substrate that produced pronounced enzyme self-inactivation during catalysis. 3,4-Dehydromyristate inactivated the oxygenase without appreciable oxygen consumption. Several compounds inhibited oxygenase activity, including catechol (Ki ~ 90 μM), divalent zinc ion (Ki ~ 50 μM), N,N,N',N'-tetramethyl-p-phenylenediamine (Ki ~20 μM) and cyanide ion (Ki ~5 μM). Zinc ion did not change the Km values for linoleate or oxygen, or the Ki value for cyanide, indicating that zinc acts at a distinct site from the other compounds. Gel-filtration chromatography revealed considerable variation in oligomeric state of recombinant PADOX1 produced in the various expression systems, but oligomeric state was not correlated with activity. Deletion of the first eight or fourteen PADOX1 residues in a NuSA-PADOX1 fusion protein led to 13 and 83% decreases in activity, respectively, indicating the N-terminal region is important for normal catalytic activity.

Original languageEnglish (US)
Pages (from-to)284-293
Number of pages10
JournalPlant Physiology and Biochemistry
Issue number5-6
StatePublished - May 2006


  • Amino terminus
  • Dioxygenase
  • Fatty acid alpha oxygenase
  • Inhibitors
  • Substrate specificity

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science


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