Substrate specificity of Rv3378c, an enzyme from Mycobacterium tuberculosis, and the inhibitory activity of the bicyclic diterpenoids against macrophage phagocytosis
Substrate specificity of Rv3378c, an enzyme from Mycobacterium tuberculosis, and the inhibitory activity of the bicyclic diterpenoids against macrophage phagocytosis
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DOI：
10.1039/c0ob00884b
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发表时间：
2011-01-01
影响因子：
3.2
通讯作者：
Hara, Takashi
中科院分区：
文献类型：
--
作者：
Hoshino, Tsutomu;Nakano, Chiaki;Hara, Takashi
结核分枝杆菌Rv3378c基因产物编码一个二萜合酶，由结核二磷酸(2)合成结核菌醇(3)、13R-异结核菌醇(4a)和13S-异结核菌醇(4b)。产品分配比例为3：4为1：1，4a-4b为1：3。对Rv3378c编码酶的底物特异性进行了检测。通过该反应合成了3种二氯二烯基二磷酸铜(CDP)(7)、ent-CDP(8)和syn-CDP(9)，产率较高(67-78%)。从7个菌株中分离得到23个铜绿醇和24个甘露醇，8个菌株中分离得到25个铜绿醇和26个甘露醇，9个菌株中分离得到27个棕榈醇和28个牡荆素A，其中23：24的比例为40：27，25：26的比例为22：50，27：28的比例为16：62。在配有手性柱的GC-MS色谱仪上分析表明，24、26和28由13R-(A)和13S-立体异构体(B)组成，其比例为：24a至24b约为1：1，26a至26b约为1：5，28a至28b约为1：19。这些产物的结构表明，3个CDP的反应方式与2个的相同。这是首次报道了天然二萜甘露醇、恩特-甘露醇和卵磷脂A的酶促合成。Rv3377c和Rv3378c基因在强毒分枝杆菌中都存在，但在强毒分枝杆菌中没有发现。我们发现3和4抑制了人巨噬细胞对调理酵母多糖颗粒的吞噬。有趣的是，3和4b共存时，抑制活性协同增强。其他与Labdane相关的二萜化合物13-16和23-28几乎没有或几乎没有抑制活性。3和4的这种协同抑制作用可能进一步有利于吞噬细胞功能的损害，这可能是结核分枝杆菌致病的原因之一。
The Rv3378c gene product from Mycobacterium tuberculosis encodes a diterpene synthase to produce tuberculosinol (3), 13R-isotuberculosinol (4a), and 13S-isotuberculosinol (4b) from tuberculosinyl diphosphate (2). The product distribution ratios are 1 : 1 for 3 to 4 and 1 : 3 for 4a to 4b. The substrate specificity of the Rv3378c-encoded enzyme was examined. The 3 labdadienyl diphosphates, copalyl diphosphate (CDP) (7), ent-CDP (8), and syn-CDP (9), underwent the conversion reaction, with good yields (67-78%). Copalol (23) and manool (24) were produced from 7, ent-copalol (25) and ent-manool (26) from 8, and syn-copalol (27) and vitexifolin A (28) from 9. The ratio of 23 to 24 was 40 : 27, that of 25: 26 was 22 : 50, and that of 27: 28 was 16 : 62. Analysis on a GC-MS chromatograph equipped with a chiral column revealed that 24, 26, and 28 consisted of a mixture of 13R-(a) and 13S-stereoisomers (b) in the following ratio: ca. 1 : 1 for 24a to 24b, ca. 1 : 5 for 26a to 26b, and ca. 1 : 19 for 28a to 28b. The structures of these products indicate that the reactions of the 3 CDPs proceeded in the same fashion as that of 2. This is the first report on the enzymatic synthesis of natural diterpenes manool, ent-manool, and vitexifolin A. Both Rv3377c and Rv3378c genes are found in virulent Mycobacterium species, but not in avirulent species. We found that 3 and 4 inhibited the phagocytosis of opsonized zymosan particles by human macrophage-like cells. Interestingly, the inhibitory activity was synergistically increased by the coexistence of 3 and 4b. Other labdane-related diterpenes, 13-16 and 23-28, had little or no inhibitory activity. This synergistic inhibition by 3 and 4 may provide further advantage to the impairment of phagocyte function, which might contribute to pathogenicity of M. tuberculosis.