Marchesini Silvia, Poirier Yves
Laboratoire de Biotechnologie Végétale, Institut d'Ecologie, Université de Lausanne, CH-1015 Lausanne, Switzerland.
J Biol Chem. 2003 Aug 29;278(35):32596-601. doi: 10.1074/jbc.M305574200. Epub 2003 Jun 20.
The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.
通过监测细菌多羟基脂肪酸合酶将β-氧化中间体3-羟基酰基辅酶A在过氧化物酶体中聚合成聚羟基脂肪酸酯的过程，分析了酿酒酵母中脂肪酸向β-氧化的通量。聚羟基脂肪酸酯的合成依赖于参与生成3-羟基酰基辅酶A的β-氧化酶酰基辅酶A氧化酶和烯酰辅酶A水合酶/3-羟基酰基辅酶A脱氢酶多功能蛋白，以及参与蛋白质导入过氧化物酶体的过氧化物酶PEX5。在含有脂肪酸的培养基中生长的野生型细胞中，聚羟基脂肪酸酯单体组成在很大程度上受外部脂肪酸性质的影响，以至于油酸产生偶数链单体，十七碳烯酸产生奇数链单体。相比之下，在以油酸或十七碳烯酸为碳源生长的过氧化物酶体3-酮硫解酶缺陷型突变体（fox3Δ0）中，以及在以葡萄糖或棉子糖为碳源生长的野生型和fox3Δ0突变体中，都合成了主要含有3-羟基辛酸、3-羟基癸酸和3-羟基十二烷酸的聚羟基脂肪酸酯，这表明用于聚羟基脂肪酸酯合成的3-羟基酰基辅酶A是通过β-氧化循环降解细胞内短链和中链脂肪酸产生的。用浅蓝菌素抑制脂肪酸生物合成可阻断细胞内脂肪酸合成聚羟基脂肪酸酯，但仍能利用细胞外脂肪酸进行聚合物生产。硫辛酸合成受影响的突变体表现出正常的聚羟基脂肪酸酯合成能力。总之，这些结果揭示了一个大量无效循环的存在，即细胞质脂肪酸生物合成途径的短链和中链中间体被导向过氧化物酶体β-氧化途径。
