Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis

Siddiqui, Masood Ahmed; Yamanaka, Aiko; Hirooka, Kazutake; Bamaba, Takeshi; Kobayashi, Akio; Imanaka, Tadayuki; Fukusaki, Eiichiro; Fujiwara, Shinsuke

doi:10.1016/j.bbrc.2005.04.029

Cited by 16 publications

(17 citation statements)

References 28 publications

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“…The spectral changes for the reduced, enzyme-bound 1-deaza and 5-deazaFMN analogues in the presence of IPP are also consistent with the accumulation of their neutral reduced forms. The protonation in the N1/C1-C2═O2′ region of all three coenzymes may be mediated by Lys186 or His149 (S. aureus numbering), both of which are absolutely conserved among IDI-2 enzymes (15)(16)(17)20). Previously, we have suggested that IPP binding may induce a conformational change that results in a K m value for reduced FMN (200 nM) that is 70-fold lower than the K d (14 µM) value for binding of the reduced FMN to IDI-2 in the absence of IPP (21).…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…such as Streptomyces and Bacillus, and by all archaea the complete genome sequences of which have been determined. [19][20][21][22][23][24][25] This protein is a flavoprotein requiring FMN, NAD(P)H, and a divalent metal for activity. Presumably, NAD(P)H is needed to reduce the flavin and generate an active IDI2-reduced flavin (FMNH 2 ) complex.…”

Section: Introductionmentioning

confidence: 99%

Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase

Dutoit¹,

Ruyck

Durisotti³

et al. 2007

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In the first step of this study, type 2 isopentenyl diphosphate isomerase (IDI2) from Pyrococcus furiosus (pf-IDI2), a hyperthermophilic microorganism, was cloned and overexpressed in E. coli. After purification, hyperthermophilic behavior of this protein was approached by means of enzymatic assays and thermal denaturation studies. Compared with the mesophilic Streptococcus pneumoniae IDI2, which unfolds and looses activity above 50 degrees C, pf-IDI2 is still folded and active at 80 degrees C. Molecular modeling was applied, in a parallel step, to understand the molecular basis of thermal stability. Comparison of IDI2 from S. pneumoniae, T. thermophilus, and P. furiosus suggested that additional charged residues present in the hyperthermophilic enzyme might contribute to its higher thermal stability. This could increase the number of salt bridges between monomers of IDI2 in P. furiosus enzyme and, hence, decrease flexibility of loops or N-terminal segment, thereby enhancing its thermal stability.

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“…The concentration of the stock solution of enzyme was detemined by UV spectrometry (16, 41) and its activity was determined by the acid lability assay. Samples wee incubated at 37 °C and NMR spectra were collected at 500 MHz.…”

Section: Methodsmentioning

confidence: 99%

Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues

2010

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Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition/abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP, but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1, but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP.

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Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis

Cited by 16 publications

References 28 publications

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

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

Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase

Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues

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