Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i>

oleh: Daniel Maynard, Kamel Chibani, Sonja Schmidtpott, Thorsten Seidel, Jens Spross, Andrea Viehhauser, Karl-Josef Dietz

Format: Article
Diterbitkan: MDPI AG 2021-09-01

Deskripsi

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated <i>A. thaliana</i> 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (V<i><sub>max</sub></i> <sub><i>app</i></sub>) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s<sup>−1</sup>·mg protein<sup>−1</sup>), with a K<sub>m</sub> value of 5.8 µM. <i>A. thaliana</i> 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg<sup>2+</sup>, Ca<sup>2+</sup>, Cu<sup>2+</sup> and Cd<sup>2+</sup>, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.