4 結核菌に見いだされたハリマン骨格の新規ジテルペン : クローニング、機能解析、基質特異性及び生物活性(口頭発表の部)

概要

論文の詳細を見る
Based upon the genomic database of Mycobacterium tuberculosis H37Rv, the gene Rv3377c,which is a putative terpene cyclase, was amplified by PCR and ligated into BamH I/Hind III site of pET 22b(+), and expressed in E. coli. BL21(DE3). The expressed Rv3377c gene product is almost insoluble (inclusion body), but coexpression of Rv3377c and chaperon in E. coli afforded the gene product as an active form. The functionally expressed gene product had an enzyme activity only for geranylgeranyl diphosphate (GGPP), but inert to geranyl-pp (GPP), farnesyl-pp (FPP), squalene and oxidosqulaene. The structural analysis of the enzymatic product revealed that this enzyme encodes the diterpene cyclase for producing the halimane diterpene skeleton. We named tuberculosinol 1 for the enzyme product. The detailed investigation on the gene product indicated that the actual product by the Rv3377c enzyme was tuberculosinol diphosphate 2. Cyclic diterpene skeletons are usually constructed after removing the PP functional group. After the trials to find the dephosphorylating enzyme, it has turned out that the flanking gene Rv3378c is responsible for the dephosphorylation reaction of 2, yielding the hydroxylated product (isotuberculosinol 3) and 1 resulting from a water attack. Both of the Rv3377c and Rv3378c have been found only the pathogenic species, but not in the non-pathogenic one. Recently, Russell and coworkers reported that the survival of Mycobacterium tuberculosis in the macrophage was attenuated by the gene disruption Thus, the two genes may be closely related to the pathogenicity. We evaluated the effect of 1 on phagocytic activity against zymosan, a yeast cell wall component that produces inflammation, using human macrophage-like U937 cells. Fluorescence microscopic analysis revealed that the phagocytosis of FITC-labeled zymosan by U937 cells treated with 10μM of 1 was reduced by about 50% compared to the cells without treatment. We discuss the detailed enzyme characterization including the kinetic data and substrate specificities. The substrate specificity of the Rv3378c enzyme was remarkably broad as well as cyc2, which was found in Streptomyces griseolosporeus. The Rv3378c and cyc2 accepted copalyl-PP (CDP), ent-CDP and syn-CDP as substrate, leading to the successful construction of a variety of diterpene skeletons.
天然有機化合物討論会の論文
2006-09-15

4 結核菌に見いだされたハリマン骨格の新規ジテルペン : クローニング、機能解析、基質特異性及び生物活性(口頭発表の部)

スポンサーリンク

概要

著者

関連論文

スポンサーリンク