龙须菜血红素加氧酶138位亮氨酸突变对藻红胆素合成的影响

Recombinant Expression of Heme Oxygenase from Gracilariopsis lemaneiformis and Its Function Analysis in Phycoerythrobilin Synthesis

藻红胆素是一个开链四吡咯结构的发色团,其与脱辅基藻红蛋白结合使藻红蛋白具有光学活性,而血红素加氧酶基因ho对于藻红胆素的合成是至关重要的。本研究对实验室前期已获得的龙须菜中的血红素加氧酶基因ho-1的基因序列进行分析发现,其在藻类中比较保守,存在6个高度保守结构域;结构功能分析显示其具有15个可能的血红素(Heme)结合位点,其中第138位亮氨酸(L)既位于118~145位HemeO的扭结螺旋结构内,又在115~142位的高度保守的结构域内,而且经预测是可能的血红素(Heme)结合位点之一。为了进一步验证该位点的作用,我们拟对其进行定点突变,将第413位碱基由T突变成C,得到ho(M)基因,碱基突变后所对应编码的第138位氨基酸由亮氨酸(L,非极性氨基酸)突变为丝氨酸(S,极性氨基酸)。在此基础上,我们构建了重组质粒pET-ho-1、pET-ho-1-pebS、pET-ho-1-pebA-pebB及pET-ho(M)、pET-ho(M)-pebS、pET-ho(M)-pebA-pebB,将其转化到E.coli BL21中构建重组菌株E.coli ph、E.coli phS、E.coli phAB、E.coli ph(M)、E.coli ph(M)S和E.coli ph(M)AB进行表达,检测重组菌株的荧光发射光谱。结果表明,E.coli phAB和E.coli phS在595 nm处检测到藻红胆素的荧光特征峰,表明ho-1基因编码的血红素加氧酶可以参与催化藻红胆素的合成;E.coli ph(M)、E.coli ph(M)S和E.coli ph(M)AB与E.coli BL21一样无任何特征峰出现,这表明ho(M)中第413位碱基对应编码的第138位亮氨酸是血红素的结合位点,直接影响藻红胆素的合成,该位点的突变使得PebS和PebB/A两条催化途径均无法合成藻红胆素。本研究为阐明血红素加氧酶基因在合成藻红胆素过程中的重要性及龙须菜中藻红蛋白的合成机制,进而进行藻红蛋白的重组表达奠定了基础。

Phycoerythrobilin is a chromophore with an open chain tetrapyrrole structure,which combines with apo-phycoerythrin to make phycoerythrin have optical activity.Heme oxygenase gene ho is crucial for the synthesis of phycoerythrobilin.In this study,the gene sequence of heme oxygenase gene ho-1 in Gracilariopsis lemaneiformis obtained in our previous work was analyzed,which was conserved in algae and 6 highly conserved domains were found by cluster analysis with closely related species.Structural and functional analysis showed that HO-1 had 15 possible Heme binding sites,among which leucine(L)at position 138 was located in the kink helix structure of HemeO at positions 118~145 and also in the highly conserved domain at positions 115~142.In order to further verify the role of this site,we intend to carry out site-directed mutation on L138 through mutation the 413th base from T to C to obtain ho(M)gene.After the base mutation,the corresponding encoded 138th amino acid is mutated from leucine(L,nonpolar amino acid)to serine(S,polar amino acid).On this basis,we constructed recombinant plasmids pET-ho-1,pET-ho-1-pebS,pET-ho-1-pebA-pebB and pET-ho(M),pET-ho(M)-pebS,pET-ho(M)-pebA-pebB.The recombinant strains E.coli ph,E.coli phS,E.coli phAB,E.coli ph(M),E.coli ph(M)S and E.coli ph(M)AB were obtained by transformation and induced to express the protein.The fluorescence emission spectra of the recombinant strains were detected,and the results showed that the fluorescence characteristic peak of phycoerythrin was detected at 595 nm in E.coli phAB and E.coli phS,which indicated that heme oxygenase encoded by ho-1 gene could involve in the synthesis of phycoerythrobilin.E.coli ph(M)S and E.coli ph(M)AB have no characteristic peaks like E.coli BL21,which indicates that the 138th leucine corresponding to the 413th base in ho(M)directly affects the synthesis of phycoerythrobilin,and the mutation of this site makes it impossible to synthesize phycoerythrobilin by PebS or PebB/A.This study laid a foundation for clarifying the importance of heme oxygenase gene in the synthesis of phycoerythrobilin and the synthesis mechanism of phycoerythrin in G.lemaneiformis,and then provided reference for recombinant expression of phycoerythrin.