The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2-sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies appear to support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.
The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steadystate and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect ( D 2 O V max = 1.5) was measured on v o in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity ( D V max = 1.8) and on the decay rate of the flavin intermediate ( D k s = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2 H]-IPP. Taken together, these data suggest that the C2-H bond of IPP is cleaved in the rate determining step and that general acid/ base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures.Isoprenoids comprise a large and ubiquitous class of metabolites that participate in numerous physiological processes (1-4). All isoprenoids are derived initially from the condensation of two isoprene units, isopentenyl pyrophosphate (IPP, 1)1 and dimethylallyl pyrophosphate (DMAPP, 2). Two biosynthetic pathways for the production of IPP and DMAPP are known, the mevalonate (MVA) pathway found in animals, fungi, and archaebacteria, and the non-mevalonate or methyl erythritol phosphate (MEP) pathway † This work was supported in part by a Welch Foundation Grant (F-1511), a National Institutes of Health Grant (GM40541), and a NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript found in most eubacteria, green algae, and the chloroplasts of higher plants (1,5,6). In the MVA pathway, DMAPP must be generated from IPP by an isopentenyl diphophate/ dimethylallyl diphosphate isomerase (IDI, Scheme 1) (1). In the MEP pathway, both IPP and DMAPP are coproduced from 4-hydroxy-3-methyl-2-butenyl diphosphate in the same enzymatic reaction (catalyzed by IspH)...
[chemical reaction: see text]. To study the catalysis of isopentenyl diphosphate (IPP) isomerase type II from Staphylococcus aureus, which is a flavoprotein catalyzing the interconversion of IPP and dimethylallyl diphosphate, we have chemically synthesized (S)- and (R)-[2-2H]IPP and carried out stereochemical analysis of the reaction. Our results show that the C-2 deprotonation of IPP by this enzyme is pro-R stereospecific, suggesting a similar stereochemical course as the type I enzyme.
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