NADPH-Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

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 omega 3 and omega 6 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 Mortierella alpina omega 3 and omega 6 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, M. alpina omega 3 and omega 6 desaturase and detergent. In this system, the maximum activity of omega 3 and omega 6 desaturase was 213.4 +/- 9.0 nmol min(-1) mg(-1) and 10.0 +/- 0.5 nmol min(-1) mg(-1), respectively. The highest k(cat)/K-m value of omega 3 and omega 6 desaturase was 0.41 mu M-1 min(-1) and 0.09 mu M-1 min(-1) when using linoleoyl CoA (18:2 omega 6) and oleoyl CoA (18:1 omega 9) as substrates, respectively. M. alpina omega 3 and omega 6 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 omega 3 and omega 6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.