Zhang Y W, Koyama T, Marecak D M, Prestwich G D, Maki Y, Ogura K
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
Biochemistry. 1998 Sep 22;37(38):13411-20. doi: 10.1021/bi972926y.
Heptaprenyl diphosphate synthase of Bacillus subtilis, which participates in the biosynthesis of the side chain of menaquinone-7, is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon [Zhang, Y.-W., et al. (1997) J. Bacteriol. 179, 1417-1419]. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg2+ and farnesyl diphosphate was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, farnesyl diphosphate, and Mg2+ form a ternary complex which seems to represent a catalytically active state of the heptaprenyl diphosphate synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg2+. A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the component I protein, suggesting that component I possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.
枯草芽孢杆菌的庚基二磷酸合酶参与维生素K7侧链的生物合成，由两个可解离的亚基组成，即组分I和组分II，它们由gerC操纵子的一个新基因簇中的两个顺反子编码[张，Y.-W.等人（1997年）《细菌学杂志》179，1417 - 1419]。这种酶的催化作用基本上需要两个亚基同时存在。分别构建了两个结构基因gerC1和gerC3的表达载体系统，并在大肠杆菌细胞中过量表达这两个组分。纯化后，通过凝胶过滤和免疫印迹分析研究了它们在形成催化活性复合物过程中的动态相互作用。当在Mg2 +和法尼基二磷酸存在下预孵育的两个组分的混合物进行Superdex 200凝胶过滤时，在一个比它们单独层析时观察到的区域更早的区域出现了一个明显的洗脱峰。这个级分包含组分I和II，其对应的分子量与两个组分的值之和一致。交联研究表明，两个必需亚基、法尼基二磷酸和Mg2 +形成了一个三元复合物，这似乎代表了庚基二磷酸合酶的催化活性状态。另一方面，在异戊烯基二磷酸或无机焦磷酸和Mg2 +存在下没有形成复合物。法尼基二磷酸的光亲和类似物被证明优先标记组分I蛋白，表明组分I对烯丙基底物具有特异性亲和力。此外，在组分II存在下，组分I的光亲和标记显著增加。通过假设两个亚基的缔合和解离促进了从可溶性底物合成两亲性产物的催化周转，来理解这种独特的异源二聚体酶的催化机制。
