NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i>
Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition o...
| المؤلفون الرئيسيون: | , , , |
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| التنسيق: | مقال |
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MDPI AG
2022-04-01
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| سلاسل: | Current Issues in Molecular Biology |
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| الوصول للمادة أونلاين: | https://www.mdpi.com/1467-3045/44/5/125 |
| _version_ | 1827669619961233408 |
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| author | Mingxuan Wang Jing Li Wenjie Cong Jianguo Zhang |
| author_facet | Mingxuan Wang Jing Li Wenjie Cong Jianguo Zhang |
| author_sort | Mingxuan Wang |
| collection | DOAJ |
| description | Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of <i>Mortierella alpina</i> ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, <i>M. alpina</i> ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min<sup>−1</sup> mg<sup>−1</sup> and 10.0 ± 0.5 nmol min<sup>−1</sup> mg<sup>−1</sup>, respectively. The highest <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value of ω3 and ω6 desaturase was 0.41 µM<sup>−1</sup> min<sup>−1</sup> and 0.09 µM<sup>−1</sup> min<sup>−1</sup> when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. <i>M. alpina</i> ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in <i>M. alpina</i>. |
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| issn | 1467-3037 1467-3045 |
| language | English |
| last_indexed | 2024-03-10T03:08:40Z |
| publishDate | 2022-04-01 |
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| series | Current Issues in Molecular Biology |
| spelling | doaj.art-62b3f058c2f745099484fd0ae75f86882023-11-23T10:32:05ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452022-04-014451828183710.3390/cimb44050125NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i>Mingxuan Wang0Jing Li1Wenjie Cong2Jianguo Zhang3Institute of Food Science and Engineering, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaInstitute of Food Science and Engineering, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaInstitute of Food Science and Engineering, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaInstitute of Food Science and Engineering, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaFatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of <i>Mortierella alpina</i> ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, <i>M. alpina</i> ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min<sup>−1</sup> mg<sup>−1</sup> and 10.0 ± 0.5 nmol min<sup>−1</sup> mg<sup>−1</sup>, respectively. The highest <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value of ω3 and ω6 desaturase was 0.41 µM<sup>−1</sup> min<sup>−1</sup> and 0.09 µM<sup>−1</sup> min<sup>−1</sup> when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. <i>M. alpina</i> ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in <i>M. alpina</i>.https://www.mdpi.com/1467-3045/44/5/125ω3 desaturaseω6 desaturaseNADPH–cytochrome P450 reductaseenzyme kinetics<i>Mortierella alpina</i> |
| spellingShingle | Mingxuan Wang Jing Li Wenjie Cong Jianguo Zhang NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i> Current Issues in Molecular Biology ω3 desaturase ω6 desaturase NADPH–cytochrome P450 reductase enzyme kinetics <i>Mortierella alpina</i> |
| title | NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i> |
| title_full | NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i> |
| title_fullStr | NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i> |
| title_full_unstemmed | NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i> |
| title_short | NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from <i>Mortierella alpina</i> |
| title_sort | nadph cytochrome p450 reductase mediates the fatty acid desaturation of ω3 and ω6 desaturases from i mortierella alpina i |
| topic | ω3 desaturase ω6 desaturase NADPH–cytochrome P450 reductase enzyme kinetics <i>Mortierella alpina</i> |
| url | https://www.mdpi.com/1467-3045/44/5/125 |
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