Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i>
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...
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MDPI AG
2021-09-01
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| Online Access: | https://www.mdpi.com/1422-0067/22/19/10237 |
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| author | Daniel Maynard Kamel Chibani Sonja Schmidtpott Thorsten Seidel Jens Spross Andrea Viehhauser Karl-Josef Dietz |
| author_facet | Daniel Maynard Kamel Chibani Sonja Schmidtpott Thorsten Seidel Jens Spross Andrea Viehhauser Karl-Josef Dietz |
| author_sort | Daniel Maynard |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T07:00:55Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T07:00:55Z |
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| spelling | doaj.art-99e2e7559adf4ba99addb0f53c67c8d92023-11-22T16:06:26ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-09-0122191023710.3390/ijms221910237Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i>Daniel Maynard0Kamel Chibani1Sonja Schmidtpott2Thorsten Seidel3Jens Spross4Andrea Viehhauser5Karl-Josef Dietz6Department of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, GermanyDepartment of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, GermanyDepartment of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, GermanyDepartment of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, GermanyFaculty of Chemistry/Industrial Organic Chemistry and Biotechnology, University of Bielefeld, 33615 Bielefeld, GermanyDepartment of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, GermanyDepartment of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, Germany13-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.https://www.mdpi.com/1422-0067/22/19/10237<i>Arabidopsis thaliana</i>lipoxygenasespolyunsaturated fatty acidslipoxygenase inhibitoroxylipin |
| spellingShingle | Daniel Maynard Kamel Chibani Sonja Schmidtpott Thorsten Seidel Jens Spross Andrea Viehhauser Karl-Josef Dietz Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i> International Journal of Molecular Sciences <i>Arabidopsis thaliana</i> lipoxygenases polyunsaturated fatty acids lipoxygenase inhibitor oxylipin |
| title | Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i> |
| title_full | Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i> |
| title_fullStr | Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i> |
| title_full_unstemmed | Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i> |
| title_short | Biochemical Characterization of 13-Lipoxygenases of <i>Arabidopsis thaliana</i> |
| title_sort | biochemical characterization of 13 lipoxygenases of i arabidopsis thaliana i |
| topic | <i>Arabidopsis thaliana</i> lipoxygenases polyunsaturated fatty acids lipoxygenase inhibitor oxylipin |
| url | https://www.mdpi.com/1422-0067/22/19/10237 |
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