|
L-cysteine從亞硫酸的生合成過程中亞硫酸的還原作用是必須的。大腸桿菌的NADPH:sulfite reductase其催化的反應為六個電子的轉移，將sulfite還原成sulfide，而特別以NADPH為電子提供者。
在定序Bacillus megaterium cytochrome P450 BM3上游核酸序列時發現有一段約250 bp 序列與 Escherichia coli及 Salmonellatyphimuriun亞硫酸還原酵素有很高相似性。隨後發現其與上下游為不連續序列。在Bacillus屬亞硫酸還原酵素還沒被找到，因此我們決定針對B.megaterium的亞硫酸還原酵素做選殖，定序與分析。
我們用上述的250 bp序列為探針篩選B.megaterium的genomic library，最後得到六個clones。將完整的序列定出後，以電腦初步分析，得到兩個相鄰的開放讀碼框，分別編碼572及573個胺基酸。在第二個開放讀碼框下游找到一個stem-loop結構，可能作為rhO-independent的轉錄終止訊號。該兩個開放讀碼框編碼蛋白經比對發現分別與E. coli的亞硫酸還原酵素的α、β次單元有很高的相似性。將含有ORF2的DNA段落送入E. COli中表現，得到與預測值約相符的60kDa。用同源性重組的方式將ORF2破壞得到B.megaterium突變株，並由南方氏墨點法確認。該突變株能在供應sulfide的minimal medium中生長但無法在sulfite的M9而minimal medium中生長。以能夠持續表現的ORF2表現質體互補ORF2突變株，可以回復其在供應sulfite的M9 minimal medium中生長。用CAT assay 分析ORF1上游區域的序列顯示了啟動子坐落位子。轉錄起始點由引子延長法分析顯示坐落在ORF1的轉譯起始點上游211 bp的位置。 6-electron reduction of sulfite to sulfide specifically by NADPH. Nucleotide sequence anaiysis of the upstream region ofcytochrome P450 BM3 gene in Bacillus megatenum revealed a region about 250 bp long, which was similar to the sulfite reductases of E. coli and Salmonella typhimurium. However, this region is discontinuous from its flanking sequences. Since no information about sulfite reductase from Bacillus spp. was available, we decided to clone, sequence, and characterize the sulfitereductase gene from B. megaterium.
The aforementioned 250-bp DNA fragment was used as a probe to screen a genomic library of B. megaterium. Six positive clones were found. Determination of the nucleotide sequences of the cloned fragments revealed two adjacent open reading frames (ORF1 and ORF2) which encode putative proteins of 572 and 573 amino acids, respectively. A stem-loop structure which may serve as a rho-independent transcription terminator was identified in the immediate downstream region of ORF2. The deduced amino acid sequences of these two ORFs are highly homologous with those of α and β subunits of sulfite reductase from E. coli. Expression of a DNA fragment containing ORF2 in E. coli resulted in the synthesis of a polypeptide of about 60 kDa, which is consistent with the predicted size. A B. megaterium mutant strain bearing disrupted ORF2 was constructed by homologous recombination and confirmed by Southern blot analysis. This mutant strain could grow on minimal medium with sulfide, but not on minimal medium with sulfite. Complementation of this ORF2 mutation with an expression vector, which constitutively expressed ORF2, restored the ability to grow on minimal medium with sulfite. Chloramphenicol acetyltransferase (CAT) analysis of the deletion derivatives of the upstream region of ORF1 revealed the location of the putative promoter(s). The transcription start site was mapped by primer extension analysis to be 211 bp upstream of the translation start site of ORF1.
|