Faculty of Biochemistry and Molecular Medicine, and Biocenter Oulu, University of Oulu, Oulu, Finland.
Theoder-Boveri-Institut für Biowissenschaften der Universität Würzburg, Würzburg, Germany.
J Struct Biol. 2020 Jun 1;210(3):107494. doi: 10.1016/j.jsb.2020.107494. Epub 2020 Mar 11.
Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli, the last three steps of the β-oxidation are catalyzed by two heterotetrameric αβ enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the β-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-β is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of β-oxidation in E. coli.
脂肪酸通过β-氧化途径降解生成乙酰辅酶 A，后者进入三羧酸循环生成 ATP。在大肠杆菌中，β-氧化的最后三步由两种异源四聚体的 αβ 酶催化，即需氧三功能酶（EcTFE）和厌氧 TFE（anEcTFE）。TFE 的 α 亚基具有 2E-烯酰辅酶 A 水合酶（ECH）和 3S-羟酰辅酶 A 脱氢酶（HAD）活性，而β 亚基是一种硫酯酶，具有 3-酮酰基辅酶 A 硫酯酶（KAT）活性。最近的研究表明，两种 TFEs 具有互补的底物特异性，使得长链脂肪酸辅酶 A 在需氧条件下完全降解为乙酰辅酶 A。此外，还表明 EcTFE 和 anEcTFE 四聚体的组装类似于假单胞菌 fragi 和人类的 TFEs。这里进一步表征了 EcTFE 亚基的特性。引人注目的是，当单独表达时，EcTFE-α是具有催化活性的单体，而 EcTFE-β是无活性的。然而，当混合在一起时，可重新构成活性 EcTFE 四聚体。还报道了 EcTFE-α链的晶体结构，与 ATP 复合，结合在其 HAD 活性位点。结构比较表明，与其他 TFE 相比，EcTFE 水合酶活性位点的脂肪酸尾部结合口袋相对较小，与其对短链 2E-烯酰辅酶 A 底物的优先特异性一致。此外，还观察到毫摩尔浓度的 ATP 使 EcTFE 复合物不稳定，这可能对大肠杆菌中 ATP 介导的β-氧化调节有影响。
