Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae
Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae
复制标题
DOI：
10.1128/jb.180.3.667-673.1998
复制
发表时间：
1998-02-01
影响因子：
3.2
通讯作者：
Doi, Y
中科院分区：
文献类型：
--
作者：
Fukui, T;Shiomi, N;Doi, Y
对嗜水气单胞菌的聚羟基链烷酸酯（PHA）阴性突变体的互补分析证明，位于PHA合酶基因下游的402-bp基因中的ORF 3参与了PHA在链烷酸上的生物合成，并且ORF 3类型在此被称为phaJ（Ac）。将phaJ（Ac）基因导入大肠杆菌BL 21（DE 3）中，在T7启动子的调控下高效表达烯酰辅酶A（enoyl-CoA）水合酶。纯化的水合酶的N-末端氨基酸序列对应于从phaJ（Ac）的核苷酸序列推导的氨基酸序列，除了起始的Met残基。由phaJ（Ac）编码的烯酰辅酶A水合酶对具有4至6个碳原子的反式-2-烯酰辅酶A表现出（R）特异性水合活性。这些结果已经证明，由phaJ（Ac）的翻译产物催化的2-烯酰-CoA的（R）特异性水合是用于将来自脂肪酸β-氧化的（R）-3-羟基酰基-CoA单体单元供应到A中的聚（3-羟基丁酸酯-共-3-羟基己酸酯）生物合成的通道途径。开玩笑。
Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 genre is here referred to as phaJ(Ac). Escherichia coli BL21(DE3) carrying phaJ(Ac) under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ(Ac) except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJ(Ac) exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ(Ac) is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid beta-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae.