An unusual isopentenyl diphosphate isomerase found in the mevalonate pathway gene cluster from Streptomyces sp. strain CL190

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 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...

Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus

“…Furthermore, they suggested that the enzyme-bound FMN intermediate that forms during the normal reaction is consistent with a neutral reduced dihydroflavin (4, Scheme 2). Although the steady-state kinetic parameters have been reported for a few IDI-2 enzymes (13,15,18,21,22,25), the nature of the rate limiting step(s) in the catalytic mechanism is unknown. In this paper, we report the investigation of the FMN intermediate formed during the catalysis of S. aureus IDI-2 using UV-vis and EPR spectroscopies.…”

“…The reactants were allowed to mix for variable lengths of time (from 3 ms to 1 s) prior to quenching with a solution of HCl/MeOH (25%). Each time point was acquired in duplicate, and the transfer of the 14 C radiolabel from IPP to DMAPP at each quenched time point was determined using a modified Satterwhite activity assay (13). Each quenched sample was immediately incubated at 37 °C for 10 min to allow conversion of the acid-labile DMAPP product into a mixture of extractable alcohols and methyl ethers.…”

“…CL190, IDI-2 enzymes have been found in many eubacteria and archaebacteria as part of either the MVA or the MEP biosynthetic pathways (13)(14)(15). Subsequent biochemical and structural characterization of IDI-2 (13,(15)(16)(17)(18)(19)(20)(21)(22) has revealed that the IDI-2-FMN ox complex is reduced with stoichiometric amounts of NAD(P)H via stereospecific transfer of the pro-S hydride and that the resultant IDI-2-FMN red is capable of performing multiple turnovers (21). Recent redox titrations of IDI-2 from Staphylococcus aureus and Thermus thermophilus in the presence and absence of IPP (1) have shown that the apoenzyme thermodynamically stabilizes the neutral flavin semiquinone in the presence of IPP (21,22).…”

“…The type I IDI (or IDI-1), which has been extensively studied, uses divalent metal ions (Mg 2+ and Zn 2+ ) to elicit an acid/base mediated 1,3-antarafacial proton addition/elimination reaction through a 3° carbocation intermediate (8)(9)(10)(11)(12). The type II IDI (IDI-2), which was discovered more recently, requires flavin mononucleotide (FMN), a reduced nicotinamide adenine dinucleotide cofactor (either NADH or NADPH), and divalent metal ions (Mg 2+ ) for catalysis (13). Since its initial isolation from Streptomyces sp.…”

Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate-Induced Flavin Species

“…Type II IPP isomerase was first reported in 2001 (15). In contrast to the type I enzyme, type II isomerase requires flavin mononucleotide (FMN), a reducing agent, typically NADPH, and a divalent metal for activity.…”