Arabidopsis thaliana fatty acid alpha-dioxygenase-1: evaluation of substrates, inhibitors and amino-terminal function.
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 (K(i) approximately 90 microM), divalent zinc ion (K(i) approximately 50 microM), N,N,N',N'-tetramethyl-p-phenylenediamine (K(i) approximately 20 microM) and cyanide ion (K(i) approximately 5 microM). Zinc ion did not change the K(m) values for linoleate or oxygen, or the K(i) 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.
Liu
W
W
Department of Internal Medicine, University of Texas Health Science Center at Houston, MSB 5.284, 6431 Fannin Street, TX 77030, USA.
Wang
L-H
LH
Fabian
P
P
Hayashi
Y
Y
McGinley
C M
CM
van der Donk
W A
WA
Kulmacz
R J
RJ
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D005227:Fatty Acids / Q000378:metabolism*; D009154:Mutation; D010101:Oxygen Consumption; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D005227:Fatty Acids; D011993:Recombinant Fusion Proteins; D010544:Peroxidases; C488669:fatty acid alpha-dioxygenase-1, Arabidopsis