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 Grampositive 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 mol͞mol. 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.I soprenoids play important roles in all living organisms; they function as steroid hormones in mammals, carotenoids in plants, and ubiquinone or menaquinone in bacteria (1). All of these isoprenoids are synthesized by consecutive condensations of the five-carbon precursor isopentenyl diphosphate (IPP) to its isomer dimethylallyl diphosphate (DMAPP). Two distinct pathways for the IPP biosynthesis are known. One is the mevalonate pathway that operates in eukaryotes, archaebacteria, and the cytosols of higher plants. The other is the nonmevalonate pathway ( Fig. 1), which is generally used by many eubacteria, including Escherichia coli and Bacillus subtilis, green algae, and the chloroplasts of higher plants (2, 3). The initial step of this pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase (4-7). In the second step, DXP is converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) by DXP reductoisomerase (8-11). MEP is then cytidylylated by MEP cytidylyltransferase to give 4-(cytidine 5Ј-diphospho)-2-Cmethyl-D-erythritol (CDP-ME) (12, 13), which is phosphorylated by CDP-ME kinase to yield 2-phospho-4-(cytidine 5Ј-diphospho)-2-C-methyl-D-erythritol (CDP-ME2P; refs. 14, 15). Next, CDP-ME2P is converted to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) by the action of MECDP synthase (16,17). However, the subsequent reactions leading to the formation of IPP from MECDP remain...
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