Inhibition of type 2 isopentenyl diphosphate isomerase from Methanocaldococcus jannaschii by a mechanism-based inhibitor of type 1 isopentenyl diphosphate isomerase

Hoshino, Takao; Tamegai, Hideyuki; Kakinuma, Katsumi; Eguchi, Tadashi

doi:10.1016/j.bmc.2006.06.011

Cited by 31 publications

(55 citation statements)

References 15 publications

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“…Thus, reduced FMN is the likely catalyst for protonation of IPP and DMAPP and for deprotonation of the 3°carbocationic intermediate. Previous work with mechanism-based inhibitors indicates that the polycyclic isoalloxazine unit in FMN red is in intimate contact with the inhibitors (12)(13)(14)(15). This result was confirmed by our X-ray studies with S. shibatae IDI-2 reacted with vIPP and oIPP.…”

Section: Resultssupporting

confidence: 88%

“…Mechanism-based inhibitors of IDI-1, which are activated by protonation of epoxide and diene moieties in these compounds, also inactivate IDI-2 (12)(13)(14)(15). The inhibitors alkylate FMN red , which indicates that the flavin is in intimate contact with the substrate during isomerization.…”

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confidence: 99%

“…We and others initially proposed that mechanism-based inhibitors alkylate FMN red at N5, based on comparisons of UV spectra for the isolated adducts with those reported in the literature for related compounds (14,15). Because UV spectra for N5 and C4a adducts are highly dependent on solvent and pH (19), we decided to conduct a more thorough study of the structures.…”

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confidence: 99%

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Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors

Nagai

Unno

Janczak

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Evidence for an unusual catalysis of protonation/deprotonation by a reduced flavin mononucleotide cofactor is presented for type-2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes isomerization of the two fundamental building blocks of isoprenoid biosynthesis, isopentenyl diphosphate and dimethylallyl diphosphate. The covalent adducts formed between irreversible mechanismbased inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor were investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of IDI-2 binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5, which had been proposed previously. In addition, the high-resolution crystal structures of IDI-2-substrate complexes and the kinetic studies of new mutants confirmed that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer. These data provide support for a mechanism where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation.enzyme structure | mechanism of action I sopentenyl diphosphate isomerase (IDI) catalyzes the interconversion between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (1). IDI activity is essential in those organisms that synthesize isoprenoid compounds from mevalonic acid and, although not essential, is found in those that synthesize isoprenoid metabolites from methylerythritol phosphate. Two convergently evolved forms of IDI, which share no sequence or structural homology, are known. Type-1 IDI (IDI-1) is found in eukarya and some bacteria, whereas type-2 IDI (IDI-2) occurs in archaea and other bacteria. There is no apparent correlation between the occurrence of the IDI isoforms and the use of mevalonate or methylerythritol phosphate in isoprenoid biosynthesis.IDI-1 catalyzes isomerization between IPP and DMAPP by a protonation-deprotonation reaction through a 3°carbocationic intermediate (1). The mechanism of the reaction and identification of essential active cysteine and glutamate residues were confirmed by inhibition studies using active-site-directed irreversible inhibitors (2-5). In contrast, IDI-2 is a flavoprotein that requires reduced FMN for activity (6, 7). UV-visible spectroscopic studies indicate that the anionic flavin in IDI-2 · FMN red − is protonated upon substrate binding to give a catalytically competent IDI-2 · FMN red · IPP complex (8). Recent studies indicated that the mechanism for the IDI-2 is similar to the protonation-deprotonation sequence used by IDI-1. These include measurement of deuterium kinetic isotopic effects in D 2 O or with (R)-[2-2 H]-IPP (9), linear free energy studies with seven-and eight-substituted FMN analogues (10), and studies with alkyne and allene analogues of IPP and DMAPP (11). In contrast, at...

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Section: Resultssupporting

confidence: 88%

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confidence: 99%

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confidence: 99%

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Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors

Nagai

Unno

Janczak

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…In this case, FMNH 2 could passively serve in a structural role (17) or could actively participate by protonating the double bond in IPP with a concomitant transient 2 e − oxidation of the cofactor. A protonation/deprotonation mechanism was recently suggested by Hoshino and coworkers (45), following the discovery that an epoxide analogue of IPP, a potent mechanism-based inhibitor of type I IPP isomerase (46), covalently inactivated the type II enzyme. However, it is not clear that the mechanisms for inactivation of the two proteins by the epoxide are similar.…”

Section: Discussionmentioning

confidence: 99%

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

2007

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Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni 2+ affinity chromatography. Aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH, dithionite, or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg 2+ dependent manner with K D ~ K m IPP , suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,Ndimethyl-3-amino-1-propyl diphosphate (NIPP), a transition state analog for the type I isomerase, competitively inhibits the type II enzyme, but with much lower affinity. pH dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UVvisible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and re-oxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.Isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules (Scheme 1). This family of natural products now consists of over 35,000 compounds (1), which comprise several classes of biologically important molecules, including sterols, carotenoids, prenylated proteins, dolichols, heme A, and ubiquinones. IPP isomerase is essential in organisms that generate IPP through the mevalonate pathway found in eukaryotes, archaea, and some gram-positive eubacteria (2). The enzyme, although not essential, is also found in most organisms that utilize the methylerythritol phosphate pathway, where isomerase activity is probably important for balancing the pools of IPP and DMAPP (3).Two structurally unrelated forms of IPP isomerase have been identified. The type I enzyme resides in eukaryotes and in some eubacteria, while the type II protein is found in archaea and other eubacteria (4). Type I IPP isomerase was discovered over 40 years ago and has been extensively characterized (5). The enzyme is a zinc metalloprotein (6) that catalyzes the isomerization of IPP and DMAPP by an antarafacial (7-9) protonation-deprotonation mechanism (10;11). Structural studies and site directed mutagenesis experiments have † This work was supported by NIH Grant GM25521. SCR was supported by NIH Postdoctoral Fellowship GM071114. *To who...

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“…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.…”

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

et al. 2008

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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)...

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Inhibition of type 2 isopentenyl diphosphate isomerase from Methanocaldococcus jannaschii by a mechanism-based inhibitor of type 1 isopentenyl diphosphate isomerase

Cited by 31 publications

References 15 publications

Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors

Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors

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

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

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