Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
Biochem Biophys Res Commun. 2020 Jul 30;528(3):453-458. doi: 10.1016/j.bbrc.2020.05.185. Epub 2020 Jun 4.
The biosynthesis of polyunsaturated fatty acids (PUFAs) in bacteria has been extensively studied. In contrast, studies of PUFA metabolism remain limited. Shewanella livingstonensis Ac10 is a psychrotrophic bacterium producing eicosapentaenoic acid (EPA), a long-chain ω-3 PUFA. This bacterium has the ability to convert exogenous docosahexaenoic acid (DHA) into EPA and incorporate both DHA and EPA into membrane phospholipids. Our previous studies revealed the importance of 2,4-dienoyl-CoA reductase in the conversion, suggesting that DHA is metabolized through a general β-oxidation pathway. Herein, to gain further insight into the conversion mechanism, we analyzed the role of acyl-CoA dehydrogenase (FadE), the first committed enzyme of the β-oxidation pathway, in DHA conversion. S. livingstonensis Ac10 has two putative FadE proteins (FadE1 and FadE2) that are highly homologous to Escherichia coli FadE. We found that FadE1 expression was induced by addition of DHA to the medium and fadE1 deletion reduced DHA conversion into EPA. Consistently, purified FadE1 exhibited dehydrogenase activity towards DHA-CoA. Moreover, its activity towards DHA- and EPA-CoAs was higher than that towards palmitoleoyl- and palmitoyl-CoAs. In contrast, fadE2 deletion did not impair DHA conversion, and purified FadE2 had higher activity towards palmitoleoyl- and palmitoyl-CoAs than towards DHA- and EPA-CoAs. These results suggest that FadE1 is the first enzyme of the β-oxidation pathway that catalyzes DHA conversion.
多不饱和脂肪酸（PUFAs）的生物合成在细菌中已经得到了广泛的研究。相比之下，PUFA 代谢的研究仍然有限。希瓦氏菌属的嗜冷菌 Ac10 是一种生产二十碳五烯酸（EPA）的细菌，EPA 是一种长链 ω-3 PUFAs。这种细菌有将外源二十二碳六烯酸（DHA）转化为 EPA 并将 DHA 和 EPA 掺入膜磷脂的能力。我们之前的研究表明，2,4-二烯酰基辅酶 A 还原酶在转化中很重要，这表明 DHA 是通过一般的β-氧化途径代谢的。在此，为了更深入地了解转化机制，我们分析了酰基辅酶 A 脱氢酶（FadE）在 DHA 转化中的作用，FadE 是β-氧化途径的第一个关键酶。希瓦氏菌属的 Ac10 有两个假定的 FadE 蛋白（FadE1 和 FadE2），它们与大肠杆菌的 FadE 高度同源。我们发现，FadE1 的表达是由 DHA 加入培养基诱导的，而 fadE1 的缺失减少了 DHA 转化为 EPA。一致地，纯化的 FadE1 对 DHA-CoA 表现出脱氢酶活性。此外，它对 DHA-和 EPA-CoAs 的活性高于对棕榈油酸酰基和棕榈酰基-CoAs 的活性。相比之下，fadE2 的缺失并没有损害 DHA 的转化，而纯化的 FadE2 对棕榈油酸酰基和棕榈酰基-CoAs 的活性比对 DHA-和 EPA-CoAs 的活性更高。这些结果表明，FadE1 是催化 DHA 转化的β-氧化途径的第一个酶。
