Active Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction
Active Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction
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DOI：
10.1021/bm301276k
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发表时间：
2012-11-01
影响因子：
6.2
通讯作者：
Doi, Yoshiharu
中科院分区：
文献类型：
--
作者：
Numata, Keiji;Motoda, Yoko;Doi, Yoshiharu
豚鼠气单胞菌的聚羟基烷酸合成酶。FA440(PhAc(Ac)，BAA21815)是最有价值的PHA合成酶之一，因为它具有合成实用生物塑料poly[(R)-3-hydroxybutytate-co-(R)-3-hydroxyhexanoate][P(3HB-co-3HHx)]的功能。然而，PHAC(Ac)的生化活性和活性中间体至今仍不清楚。在本研究中，我们克隆了PHAC(Ac)的代理体，并通过细胞内蛋白的表达实现了其高表达。系统。为了根据PHAc(Ac)的流体力学直径和比活性来确定PHAc(Ac)的活性中间体，对其聚合活性和齐聚行为进行了表征。考察了底物(R)-3-羟基丁酰辅酶A(3HB-CoA)对PHAc(Ac)(7.5um M)齐聚反应的影响，测定了希尔系数(n=2.6+/-0.4)和微观解离常数(K-m=77+/-5um M)。结果表明，PHAc(Ac)的活性中间体是含有3HB-CoA的二聚体PHAc(Ac)，作为其二聚反应的活化剂。这些信息对于揭示其二聚化与PHA合成中的功能之间的关系是至关重要的。
Polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae. FA440 (PhaC(Ac), BAA21815) is one of the most valuable PHA synthase, because of its function to synthesize a. practical bioplastic, poly[(R)-3-hydroxybutytate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. However, biochemical activity and active intermediates of PhaC(Ac) have not been clarified until now. In the present study, agency of PhaC(Ac) was cloned and overexpressed by a cell free protein expression. system. Both the polymerization activity and oligomerization behavior of the purified PhaC(Ac) were characterized in order to clarify the active intermediates of PhaC(Ac) based on the hydrodynamic diameters and specific activities of PhaC(Ac). The influences of a substrate, (R)-3-hydroxybutyryl-CoA (3HB-CoA), on the oligomerization of PhaC(Ac) (7.5 mu M), were also investigated, and then the Hill coefficient (n = 2.6 +/- 0.4) and the microscopic dissociation constant (K-m = 77 +/- 5 mu M) were determined. Based on the results, the active intermediate of PhaC(Ac) was concluded to be the dimeric PhaC(Ac) containing 3HB-CoA, as an activator for its dimerization. This information is critical for revealing the relationships between its dimerization and function in PHA synthesis.