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Comparative Study
, 98 (3), 932-7

An Unusual Isopentenyl Diphosphate Isomerase Found in the Mevalonate Pathway Gene Cluster From Streptomyces Sp. Strain CL190

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Comparative Study

An Unusual Isopentenyl Diphosphate Isomerase Found in the Mevalonate Pathway Gene Cluster From Streptomyces Sp. Strain CL190

K Kaneda et al. Proc Natl Acad Sci U S A.

Abstract

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.

Figures

Figure 1
Figure 1
IPP biosynthetic pathway in the E. coli transformants. The subsequent reactions leading to the formation of IPP or DMAPP from MECDP remain to be elucidated. X, unidentified biosynthetic intermediate of the nonmevalonate pathway.
Figure 2
Figure 2
Expression and purification of the recombinant orfD product. (A) SDS/PAGE of the enzyme. Lanes: 1, low molecular mass markers; 2, the pellet of the extract from E. coli M15 (pREP4, pQCLD41) culture after induction with isopropyl β-d-thiogalactopyranoside (IPTG); 3, the supernatant of the same extract; 4, proteins passed through an Ni-nitrilotriacetic acid agarose column; 5, the fraction eluted with 50 mM imidazole; 6, purified enzyme (1 μg) eluted with 200 mM imidazole. (B) Native PAGE of the purified enzyme. Lanes: 1, high molecular mass markers; 2, purified enzyme (0.7 μg).
Figure 3
Figure 3
Absorption spectrum of the recombinant orfD product reconstituted with FMN. A fraction containing the highest concentration of protein (2.8 mg/ml) is shown.
Figure 4
Figure 4
1H NMR spectra of the reaction mixtures. The reaction buffer contained 5 mM MgCl2, 1 mM DTT, 10 μM FMN, 2.5 mM NADPH, and 5 mM IPP. The spectra were obtained after incubation for 12 h at 37°C without (B) and with (A) the orfD product (1 mg). Small signals in ≈3.9–4.8 ppm can be ascribed to NADPH.
Figure 5
Figure 5
Multiple alignment of the amino acid sequences of orfD and other orfD homologs. Identical amino acids among six proteins are marked by asterisks. Dashes indicate gaps introduced for the optimization of the alignment. The thick lines represent glycine-rich motifs. orfD, Streptomyces sp. strain CL190; Erwi, E. herbicola Eho10 (accession no. Q01335); Syne, Synechocystis sp. strain PCC6803 (accession no. P74287); Saur, S. aureus ATCC25923; Enfa, E. faecalis (http://www.tigr.org/); Meta, M. jannaschii (accession no. Q58272); Sulf, S. solfataricus (accession no. P95997).

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