PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha
PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha
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DOI：
10.1128/aem.02935-13
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发表时间：
2013-11
影响因子：
4.4
通讯作者：
Daniel Pfeiffer;D. Jendrossek
中科院分区：
文献类型：
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作者：
Daniel Pfeiffer;D. Jendrossek
摘要聚3-羟基丁酸酯（PHB）合成酶（PhaC 1）是真养罗尔斯通氏菌（Ralstonia eutropha）和其它聚PHB细菌合成PHB的关键酶，催化3-羟基丁酰辅酶A聚合成PHB。活性测定R.真养型PHB合酶的特征在于存在滞后期和低比活性。假设滞后期是由通过未知的引发过程将无活性PhaC 1单体转化为活性二聚体形式所需的时间引起的。可通过加入非离子去污剂如hecameg [6-O-（N-庚基-氨基甲酰基）-甲基-α-d-吡喃葡萄糖苷]来减少滞后期，这明显加速了PhaCl二聚体的形成。我们鉴定了PHB颗粒相关蛋白（PGAP）PhaM作为PHB合酶活性的天然引物（激活剂）。PhaM是近年来发现的一种新型PGAP，在PHB代谢中具有多种功能。将PhaM添加到PHB合酶测定中导致具有高比活性的3 HB辅酶A的立即聚合，并且没有显著的滞后期。PhaM对（i）PhaC 1活性、（ii）PhaC 1的寡聚化、（iii）与PhaC 1的复合物形成以及（iv）体外和体内的PHB颗粒形成的影响通过纯化蛋白（PhaM、PhaC 1）与戊二醛的交联实验、通过尺寸排阻色谱法以及通过荧光显微镜检测从头合成的PHB颗粒来显示。
ABSTRACT Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-d-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.