Kaneda K, Kuzuyama T, Takagi M, Hayakawa Y, Seto H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):932-7. doi: 10.1073/pnas.98.3.932. Epub 2001 Jan 23.
A gene cluster encoding five enzymes of the mevalonate pathway had been cloned from Streptomyces sp. strain CL190. This gene cluster contained an additional ORF, orfD, encoding an unknown protein that was detected in some archaebacteria and some Gram-positive bacteria including Staphylococcus aureus. The recombinant product of orfD was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and 155 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a tetramer. The purified enzyme contained flavin mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6 mol/mol. The enzyme catalyzed the isomerization of isopentenyl diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the presence of both FMN and NADPH. The Escherichia coli plasmid expressing orfD could complement the disrupted IPP isomerase gene in E. coli. These results indicate that orfD encodes an unusual IPP isomerase showing no sequence similarity to those of IPP isomerases identified to date. Based on the difference in enzymatic properties, we classify the IPP isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent enzymes, and type 1 for the others. In view of the critical role of this isomerase in S. aureus and of the different enzymatic properties of mammalian (type 1) and S. aureus (type 2) isomerases, this unusual enzyme is considered to be a suitable molecular target for the screening of antibacterial drugs specific to S. aureus.
已从链霉菌属菌株CL190中克隆出一个编码甲羟戊酸途径五种酶的基因簇。该基因簇包含一个额外的开放阅读框orfD，其编码一种未知蛋白质，在一些古细菌和一些革兰氏阳性菌（包括金黄色葡萄球菌）中被检测到。orfD的重组产物被纯化为可溶性蛋白质并进行了表征。通过SDS-聚丙烯酰胺凝胶电泳估计该酶的分子量为37 kDa，通过凝胶过滤色谱法估计为155 kDa，这表明该酶很可能是四聚体。纯化的酶含有黄素单核苷酸（FMN），每个四聚体的含量为1.4至1.6 mol/mol。该酶在FMN和NADPH存在的情况下催化异戊烯基二磷酸（IPP）异构化生成二甲基烯丙基二磷酸（DMAPP）。表达orfD的大肠杆菌质粒可以互补大肠杆菌中被破坏的IPP异构酶基因。这些结果表明，orfD编码一种不寻常的IPP异构酶，与迄今鉴定的IPP异构酶没有序列相似性。基于酶学性质的差异，我们将IPP异构酶分为两种类型：依赖FMN和NAD(P)H的2型酶，以及其他的1型酶。鉴于这种异构酶在金黄色葡萄球菌中的关键作用以及哺乳动物（1型）和金黄色葡萄球菌（2型）异构酶不同的酶学性质，这种不寻常的酶被认为是筛选针对金黄色葡萄球菌的抗菌药物的合适分子靶点。
