Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
J Bacteriol. 2013 Jul;195(14):3213-23. doi: 10.1128/JB.00358-13. Epub 2013 May 10.
Degradation of poly(3-hydroxybutyrate) (PHB) by the thiolytic activity of the PHB depolymerase PhaZ1 from Ralstonia eutropha H16 was analyzed in the presence of different phasins. An Escherichia coli strain was constructed that harbored the genes for PHB synthesis (phaCAB), the phasin PhaP1, and the PHB depolymerase PhaZ1. PHB was isolated in the native form (nPHB) from this recombinant E. coli strain, and the in vitro degradation of the polyester was examined. Degradation resulted in the formation of the expected 3-hydroxybutyryl coenzyme A (3HB-CoA) and in the formation of a second product, which occurred in significantly higher concentrations than 3HB-CoA. This second product was identified by liquid chromatography mass spectrometry (LC-MS) as crotonyl-CoA. Replacement of PhaP1 by PhaP2 or PhaP4 resulted in a lower degradation rate, whereas the absence of the phasins prevented the degradation of nPHB by the PHB depolymerase PhaZ1 almost completely. In addition, the in vitro degradation of nPHB granules isolated from R. eutropha H16 (wild type) and from the R. eutropha ΔphaP1 and ΔphaP1-4 deletion mutants was examined. In contrast to the results obtained with nPHB granules isolated from E. coli, degradation of nPHB granules isolated from the wild type of R. eutropha yielded high concentrations of 3HB-CoA and low concentrations of crotonyl-CoA. The degradation of nPHB granules isolated from the ΔphaP1 and ΔphaP1-4 deletion mutants of R. eutropha was significantly reduced in comparison to that of nPHB granules isolated from wild-type R. eutropha. Stereochemical analyses of 3HB-CoA revealed that the (R) stereoisomer was collected after degradation of granules isolated from E. coli, whereas the (S) stereoisomer was collected after degradation of granules isolated from R. eutropha. Based on these results, a newly observed mechanism in the degradation pathway for PHB in R. eutropha is proposed which is connected by crotonyl-CoA to the β-oxidation cycle. According to this model, the NADPH-dependent synthesis of PHB with (R)-3HB-CoA as the intermediate and the PHB degradation yielding (S)-3HB-CoA, which is further converted in an NAD-dependent reaction, are separated.
聚 3-羟基丁酸酯 (PHB) 的降解由恶臭假单胞菌 H16 的 PHB 解聚酶 PhaZ1 的硫解活性分析，存在不同的相蛋白。构建了一种含有 PHB 合成基因（phaCAB）、相蛋白 PhaP1 和 PHB 解聚酶 PhaZ1 的大肠杆菌菌株。从该重组大肠杆菌菌株中以天然形式（nPHB）分离 PHB，并检查聚酯的体外降解。降解导致预期的 3-羟基丁酰辅酶 A（3HB-CoA）和第二种产物的形成，该产物以明显高于 3HB-CoA 的浓度形成。通过液相色谱-质谱联用（LC-MS）将第二种产物鉴定为巴豆酰辅酶 A。PhaP1 被 PhaP2 或 PhaP4 取代会导致降解速率降低，而没有相蛋白会使 PHB 解聚酶 PhaZ1 几乎完全不能降解 nPHB。此外，还检查了从恶臭假单胞菌 H16（野生型）和恶臭假单胞菌ΔphaP1 和ΔphaP1-4 缺失突变体中分离的 nPHB 颗粒的体外降解。与从大肠杆菌中分离得到的 nPHB 颗粒的结果相反，从恶臭假单胞菌野生型中分离得到的 nPHB 颗粒的降解产生了高浓度的 3HB-CoA 和低浓度的巴豆酰辅酶 A。与从恶臭假单胞菌野生型中分离得到的 nPHB 颗粒相比，从恶臭假单胞菌ΔphaP1 和ΔphaP1-4 缺失突变体中分离得到的 nPHB 颗粒的降解明显减少。对 3HB-CoA 的立体化学分析表明，从大肠杆菌中分离得到的颗粒降解后收集到（R）立体异构体，而从恶臭假单胞菌中分离得到的颗粒降解后收集到（S）立体异构体。基于这些结果，提出了在恶臭假单胞菌中 PHB 降解途径中观察到的新机制，该机制通过巴豆酰辅酶 A 与β-氧化循环相连。根据该模型，以（R）-3HB-CoA 为中间体的 NADPH 依赖性 PHB 合成和产生（S）-3HB-CoA 的 PHB 降解，（S）-3HB-CoA 进一步在 NAD 依赖性反应中转化，被分离。
