Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), CH-9014 St, Gallen, Switzerland.
BMC Microbiol. 2010 Oct 11;10:254. doi: 10.1186/1471-2180-10-254.
Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.
Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.
Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.
中链长度（mcl-）聚羟基烷酸酯（PHA）是许多细菌在细胞质中合成的作为能量和碳的储存化合物。PHA 代谢的关键酶是 PHA 聚合酶（PhaC）和解聚酶（PhaZ）。关于 mcl-PHA 的积累和降解是如何控制的知之甚少。有人认为，总体 PHA 代谢受β-氧化途径调控，该途径的通量由细胞内 [NADH]/[NAD]和 [乙酰-CoA]/[CoA]的比值决定。另一个控制水平可能与 PhaC 和 PhaZ 活性的调节有关。为了研究后者，需要在粗细胞提取物中进行 PhaC 和 PhaZ 的体外活性测定。
开发了两种体外测定法，可在恶臭假单胞菌 U 的粗细胞提取物中测量 PhaC 和 PhaZ 的活性。使用该测定法，证明 PhaC 的活性在氮限制培养基和辛酸上的指数生长时降低了 5 倍。相比之下，PhaZ 的活性在生长过程中仅增加了 1.5 倍。PhaC 和 PhaZ 酶活性变化的一个原因可能与与 PHA 颗粒表面蛋白的相互作用的变化有关。分离的 PHA 颗粒的 SDS-PAGE 分析表明，在生长过程中，[phasins]/[PHA]的比值降低。此外，还发现从颗粒中去除 phasins（PhaF 和 PhaI）后，PhaC 活性进一步降低。
使用本研究中开发的测定法，我们在生长过程中跟踪 PhaC 和 PhaZ 的酶活性，并将其与 PHA 颗粒上的 phasins 数量相关联。结果发现，在恶臭假单胞菌中，PhaC 和 PhaZ 同时活跃，导致 PHA 的平行合成和降解。此外，发现 PhaC 活性降低，而 PhaZ 活性在生长过程中增加。PHA 颗粒上 phasins 的可用性影响 PhaC 的活性。
