Ohnuma S, Nakazawa T, Hemmi H, Hallberg A M, Koyama T, Ogura K, Nishino T
Department of Biochemistry and Engineering, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-77 Japan.
J Biol Chem. 1996 Apr 26;271(17):10087-95. doi: 10.1074/jbc.271.17.10087.
Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.
异戊烯基转移酶催化异戊烯基二磷酸（IPP）与烯丙基二磷酸的连续缩合反应，生成链长完全由每种酶决定的异戊烯基二磷酸。为了研究连续反应机制以及最终链长的决定机制，我们设计了一种随机诱变方法。嗜热脂肪芽孢杆菌的法尼基二磷酸（FPP）合酶基因经亚硝酸钠处理进行随机诱变，以构建高拷贝质粒上的突变FPP合酶基因文库。利用类胡萝卜素合成基因、八氢番茄红素合酶和八氢番茄红素去饱和酶，通过红白筛选法（大沼，S.-i.，铃木，M.，和西野，T.（1994年）《生物化学杂志》268，14792 - 14797）从文库中筛选出具有香叶基香叶基二磷酸（GGPP）合酶活性的突变体，以在体内可视化GGPP的形成。从约24,300个突变体中鉴定出11个红色阳性克隆，并对其中4个（突变体1、2、3和4）进行酶活性分析。体外测定结果表明，所有这些突变体均产生（全 - E） - GGPP，尽管产量不同。发现每个突变体都含有一些氨基酸替换：突变体1为Y81H和L275S；突变体2为L34V和R59Q；突变体3为V157A和H182Y；突变体4为Y81H、P239R和A265T。定点诱变表明，Y81H、L34V或V157A对GGPP合酶活性的表达至关重要。特别是，将酪氨酸81替换为组氨酸最为有效，因为突变体1和4中GGPP与FPP的产量比最大。基于二级结构预测，发现酪氨酸81位于距第一个DDXXD基序约12埃的位置11处，其距离与FPP的烃基部分长度相似。这些数据可能表明，酪氨酸81的芳香环阻碍了比FPP更长的链延伸。突变酶和野生型酶动力学参数的比较揭示了一些可能与最终链长变化相关的现象。它们包括总反应速率降低、对二甲烯丙基二磷酸的Km增加、对二甲烯丙基二磷酸的Vmax降低以及对IPP的Km对烯丙基底物的依赖性。
