葛仙米血红素氧合酶基因Ns-HO1异源表达及其蛋白结构模拟

为明确葛仙米(Nostoc sphaeroides)中血红素氧合酶(Heme oxygenase, HO)基因编码蛋白的分子进化地位和高级结构, 克隆该基因并进行原核细胞表达, 氨基酸序列比对与分子进化分析和Swiss-model高级结构模拟。结果显示: 葛仙米血红素氧合酶基因Ns-HO1密码区全长为678 bp, 预编码一含225个氨基酸的蛋白质; Ns-HO1与同源蛋白氨基酸相似性达90.3%, 存在少部分位点替换突变, 但底物血红素的预结合位点(如Arg10和Tyr125)及金属离子结合位点(His17)等关键位点的氨基酸保持稳定。 构建表达载体, 葛仙米Ns-HO1在大肠杆菌中成功诱导表达, SDS-PAGE检测目的蛋白大小近26.0 kD; 分子进化树分析显示Ns-HO1与普通念珠藻、发状念珠藻中同源蛋白聚类于同一分支, 并与点状念珠藻中同源蛋白具有共同祖先。Ns-HO1蛋白多肽链形成8个主要α-螺旋，其中N和C末端的两个α-螺旋与第四螺旋共同为Ns-HO1高级结构提供底面支撑, 其他螺旋围绕该底面进一步延展并充填其中, 形成Ns-HO1分子的类夹心式结构。在高级结构中, 含血红素预结合位点氨基酸的几个螺旋(第一、 五和七螺旋)位于Ns-HO1分子外围, 这些分支螺旋为形成Ns-HO1夹心空间以利于底物血红素锚定和产物及时释放提供了条件。总之, 研究为深入了解与运用葛仙米血红素氧合酶基因的生物学功能和资源提供了基础。

THE HETEROLOGICAL EXPRESSION AND PROTEIN ADVANCED STRUCTURE MODEL OF THE HEME OXYGENASE GENE NS-HO1 IN NOSTOC SPHAEROIDES

Nostoc sphaeroides is well-known for its edible and medical characteristics in cyanobacteria, particularly such as those produced in Enshi, Hubei Province. However, in pace with the changes in times and environmental condition, further development of the resource is required based on special gene or molecular activities, such as the heme oxygenase, HO1, which catalyzes heme degradation and positively roles in tissues by reducing oxidative injure, inhibiting apoptosis and so on. But in N. sphaeroides, the gene for encoding HO1 has not been clear up to today. Here, the cloning of HO1 (heme oxygenase) gene from N. sphaeroides, and prokaryotic expression the protein, analysis and alignment of amino acid sequences and molecular evolution, structural simulation by Swiss-model advanced have been perfused in order to clarify the molecular evolutionary status and advanced structure of the gene coding protein. The PCR results showed that the coding region of the gene Ns-HO1 was 678 bp, encoding a protein of 225 amino acids; sequence analysis indicated that amino acid similarity between Ns-HO1 and the homologous proteins reached 90.3%. Amino acid sequence alignment showed that there were a few replacement mutations in the amino acids, but the prebinding sites (e.g., Arg10, Tyr125) for the binding sites of the enzymatic substrate heme and metal ion (His17) and other key sites were maintained. A recombinant expression vector was constructed, and the Ns-HO1 gene was successfully induced in E. coli, SDS-PAGE detected the target protein at 26.0 kD in MW size; molecular phylogenic tree showed that Ns-HO1 was clustered in the common clade with the homologous protein from the N. flagelliforme and N. commune, and they all shared the closest common ancestor with the homologous protein from N. punctiforme. The Ns-HO1 protein polypeptide chains can form eight main α- helices, in which the two α- helices at the end of the N and C terminus together with the fourth helix provided the bottom support for the advanced structure, and other helices extend and fill the bottom surface to form a sandwich-like structure of the Ns-HO1 molecule. In its advanced structure, the helixes (first, fifth and seventh helix) formed by several peptide segments containing the amino acids of the heme prebinding site is located on the periphery of the Ns-HO1 molecule; by side view, these branch helices provide important conditions for the formation of Ns-HO1 sandwich space for substrate heme anchoring and timely releasing of catalyzed products. This study provides a basis for further understanding and application of the biological functions and resources of heme oxygenase gene Ns-HO1 of N. sphaeroides.