Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.
Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.
复制标题
从基因组 DNA 序列推导出的转羧酶 5 S 亚基的一级结构。
DOI：
10.1016/0014-5793(93)80271-u
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发表时间：
1993
期刊：
影响因子：
3.5
通讯作者：
Samols,D
中科院分区：
文献类型：
--
作者：
Thornton,CG;Kumar,GK;Shenoy,BC;Haase,FC;Phillips,NF;Park,VM;Magner,WJ;Hejlik,DP;Wood,HG;Samols,D
舍尔马尼丙酸杆菌转羧酶是一种复杂的含有生物素的酶，由三种不同类型的30个多肽组成。它由六个二聚体外部亚基组成，通过12个生物素亚基与一个中心圆柱形六聚体亚基相连；中心六角体的每个面都有三个外部亚基。每个外部二聚体被称为5-S亚基，它与两个生物素亚基结合。该酶催化两步反应，其中甲基丙二酰辅酶A和丙酮酸形成丙酰辅酶A和草乙酸酯，5 S亚基专门催化其中一个反应。我们报道了S 5亚基单体的克隆、测序和表达。通过将分离的5个S多肽的氨基酸序列与克隆的P.shermani基因组片段上推导的开放阅读框序列进行比对，发现该基因包含编码1.3 S生物素亚基的基因。克隆的S基因编码一个519个氨基酸的蛋白质，Mr为57,793。推导出的序列显示了与丙酮酸羧基酶和草酸乙酸酯脱羧酶的广泛同源性，这两种酶催化相同或相反的反应。将一个片段定向亚克隆到pUC19中，使5 S开放阅读框能够从载体的lac启动子中得到表达。从这些细胞制备的粗提物在Western blotting上有一条免疫反应带，它与真品5 S共迁移，并完全催化5 S的部分反应。我们已克隆、测序并表达了S 5亚基的单体，表达产物具有催化活性。
Transcarboxylase fromPropionibacterium shermaniiis a complex biotin-containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and pyruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a clonedP.shermaniigenomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids,Mr, 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots which co-migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.