Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.
Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.
复制标题
血红素生物合成操作对大肠杆菌中玻璃颤菌血红蛋白不溶性的影响。
DOI：
10.1128/aem.60.7.2431-2437.1994
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发表时间：
1994
影响因子：
4.4
通讯作者：
Bailey,JE
中科院分区：
文献类型：
--
作者：
Hart,RA;Kallio,PT;Bailey,JE
在大肠杆菌中，玻璃体颤菌血红蛋白（VHb）通过其原生启动子在puc19衍生的质粒上表达时，以可溶性和不溶性形式大量积累。原子吸收光谱和电子顺磁共振光谱检测表明，不溶性形态均缺乏血红素假体基（apoVHb）。纯化的可溶性形式含有血红素（holoVHb），在光谱上与玻璃体振荡细胞产生的holoVHb难以区分。这一观察结果表明，apoVHb的不溶性可能与血红素的生物合成有关。为了验证这种可能性，研究人员进行了一系列实验，以化学和基因方式操纵血红素生物合成中的关键中间体5-氨基乙酰丙酸（ALA）的形成和转化。化学扰动包括在生长培养基中补充中间的ALA和竞争性抑制剂乙酰丙酸，乙酰丙酸可以自由地穿过细胞屏障。基因操作包括增加ALA合成酶和ALA脱水酶的基因剂量。左丙酸和ALA的补充结果表明，可溶性holoVHb水平与血红素水平相关，而不溶性apoovhb水平与血红素水平无关。可溶性与不溶性VHb的比值也与累积的总VHb水平无关。ALA合成酶和ALA脱水酶基因剂量的扩增作用是复杂的，可能涉及次要因素。结果表明，过量产生VHb的细胞中血红素生物合成的限速步骤不在于ALA的合成，而在野生型大肠杆菌中则有报道（S. Hino和A. Ishida, Enzyme, 1973:42-49）。
Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).