Takagi Motoki, Kaneda Kazuhide, Shimizu Tomohiro, Hayakawa Yoichi, Seto Haruo, Kuzuyama Tomohisa
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Biosci Biotechnol Biochem. 2004 Jan;68(1):132-7. doi: 10.1271/bbb.68.132.
We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.
我们之前将链霉菌属菌株CL190的fni基因鉴定为2型异戊烯基二磷酸（IPP）异构酶，该酶的酶活性需要黄素单核苷酸（FMN）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）两者。在枯草芽孢杆菌基因组数据库中检测到一个fni基因同源物ypgA。然而，ypgA产物的N端比链霉菌fni基因产物短约140个氨基酸。数据库搜索在ypgA基因原始起始密码子上游129、225和411个碱基处发现了三个新的推定起始密码子。最长的基因产物被命名为ypgA3，它与链霉菌fni基因产物显示出最显著的同源性。ypgA3基因在大肠杆菌中以N端带有组氨酸标签的形式表达，并对纯化的可溶性蛋白进行了详细表征。ypgA3蛋白在FMN和NADPH两者存在的情况下将IPP转化为其异构体二甲基烯丙基二磷酸。该酶在两种辅因子存在的情况下也催化逆反应。ypgA3基因的破坏对枯草芽孢杆菌并非致命。这些结果表明，枯草芽孢杆菌的ypgA3基因是fni，一个编码2型IPP异构酶的非必需基因。
