Borge G I, Vogt G, Nilsson A
MATFORSK, Norwegian Food Research Institute, As.
Lipids. 1999 Jul;34(7):661-73. doi: 10.1007/s11745-999-0411-7.
Fatty acid alpha-oxidation is an essential metabolic pathway both in plants and in mammals which is still not completely understood. We previously described and purified an alpha-oxidation enzyme in cucumber which has been used in the present investigation of the alpha-oxidation reaction mechanism. Free fatty acids, and not the CoA thioesters, were found to undergo alpha-oxidation in cucumber. 2-Hydroxy- and 2-oxopalmitic acids were identified as palmitic acid alpha-oxidation intermediates by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis in cucumber subcellular 150,000 x g(max) pellets obtained by differential centrifugation. Incubation of purified alpha-oxidation enzyme with [1-14C]palmitic acid resulted in the formation of both the above-described intermediates and the Cn-1 product, pentadecanal, and 14CO2. Besides 14CO2, 14C-formate was identified as an alpha-oxidation product from [1-14C]palmitic acid in cucumber subcellular fractions. Fe2+ stimulated the 14CO2 and 14C-formate production, and the addition of ascorbate and 2-oxoglutarate together with Fe2+ resulted in optimal alpha-oxidation activities, suggesting a dioxygenase reaction mechanism, as previously shown in mammals. NADPH and, to a lesser extent, NADH stimulated the total 14C-formate plus 14CO2 production but had only slight or no effects on 14CO2 production. H2O2 showed concentration-dependent inhibitory effects, while FAD had neither effect on 14CO2 nor 14CO2 plus 14C-formate production. The results in the present study demonstrate that an alpha-oxidation enzyme in cucumber is capable of oxidizing palmitic acid via 2-hydroxy- and 2-oxo-palmitic acid to produce pentadecanal and CO2. In contrast to the subcellular 150,000 x g(max) fraction, the purified alpha-oxidation enzyme could neither produce formate nor convert 14C-formate into 14C02, indicating two possible alpha-oxidation routes in cucumber.
脂肪酸α-氧化是植物和哺乳动物中一种重要的代谢途径，但目前仍未被完全理解。我们之前在黄瓜中描述并纯化了一种α-氧化酶，该酶已被用于本研究中对α-氧化反应机制的研究。在黄瓜中发现，进行α-氧化的是游离脂肪酸，而非辅酶A硫酯。通过差速离心获得的黄瓜亚细胞150,000×g（最大值）沉淀中，经高效液相色谱和气相色谱-质谱分析，鉴定出2-羟基棕榈酸和2-氧代棕榈酸为棕榈酸α-氧化的中间体。将纯化的α-氧化酶与[1-¹⁴C]棕榈酸一起孵育，会生成上述中间体以及Cn-1产物十五醛和¹⁴CO₂。除了¹⁴CO₂，在黄瓜亚细胞组分中，¹⁴C-甲酸也被鉴定为[1-¹⁴C]棕榈酸的α-氧化产物。Fe²⁺刺激了¹⁴CO₂和¹⁴C-甲酸的生成，同时加入抗坏血酸和2-氧代戊二酸以及Fe²⁺可产生最佳的α-氧化活性，这表明其反应机制为双加氧酶反应机制，正如之前在哺乳动物中所显示的那样。NADPH以及在较小程度上的NADH刺激了¹⁴C-甲酸和¹⁴CO₂的总生成量，但对¹⁴CO₂的生成仅有轻微影响或无影响。H₂O₂呈现出浓度依赖性抑制作用，而FAD对¹⁴CO₂以及¹⁴CO₂和¹⁴C-甲酸的生成均无影响。本研究结果表明，黄瓜中的一种α-氧化酶能够通过2-羟基棕榈酸和2-氧代棕榈酸氧化棕榈酸，生成十五醛和CO₂。与亚细胞150,000×g（最大值）组分不同，纯化的α-氧化酶既不能产生甲酸，也不能将¹⁴C-甲酸转化为¹⁴CO₂，这表明黄瓜中存在两种可能的α-氧化途径。
