Characterization of the Ry3378c Gene Product, a New Diterpene Synthase for Producing Tuberculosinol and (13R, S)-Isotuberculosinol (Nosyberkol), from the Mycobacterium tuberculosis H37Rv Genome
Characterization of the Ry3378c Gene Product, a New Diterpene Synthase for Producing Tuberculosinol and (13R, S)-Isotuberculosinol (Nosyberkol), from the Mycobacterium tuberculosis H37Rv Genome
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DOI：
10.1271/bbb.100570
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发表时间：
2011-01-01
影响因子：
1.6
通讯作者：
Hoshino, Tsutomu
中科院分区：
文献类型：
--
作者：
Nakano, Chiaki;Ootsuka, Takahiro;Hoshino, Tsutomu
来自结核分枝杆菌的Rv 3377 c和Rv 3378 c基因特异性地存在于毒性分枝杆菌物种中，但不存在于无毒力物种中。Rv 3378 c编码的酶从二磷酸结核菌素酯3产生结核菌素醇2（5（6），13（14）-halimadiene-15-ol）、13 R-5a和13 S-异结核菌素醇5 b（5（6），14（15）-halimadiene-13-ol）作为其酶促产物，表明Rv 3378 c酶在释放二磷酸部分后催化水分子的亲核加成。三种酶产物2、5a和5 b的产生与N-和C-末端His-标记的Rv 3378 c酶和麦芽糖结合蛋白融合酶无关;酶之间的产物分布比相同，对于2：5为1：1，对于5a：5 b为1：3。通过手性HPLC柱成功分离5a和5 b提供了5a和5 b的H-1-和C-13-NMR数据的首次完整归属。酶的机制产生2，5a，和5 b的建议，在这里，和最佳的催化条件和动力学参数，除了二价金属的影响，进行了描述。定点突变的天冬氨酸到天冬酰胺，针对DDXXD基序，导致酶活性显着降低。
The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of H-1- and C-13-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.