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Dale C Poulter - One of the best experts on this subject based on the ideXlab platform.
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mechanistic studies of the protonation deprotonation reactions for type 1 and type 2 Isopentenyl Diphosphate dimethylallyl Diphosphate isomerase
Journal of the American Chemical Society, 2018Co-Authors: Syam Sundar Neti, Dale C PoulterAbstract:Type 1 and type 2 Isopentenyl Diphosphate:dimethylallyl Diphosphate isomerase (IDI-1 and IDI-2) catalyze the interconversion of Isopentenyl Diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP), the fundamental building blocks for biosynthesis of isoprenoid compounds. Previous studies indicate that both isoforms of IDI catalyze isomerization by a protonation–deprotonation mechanism. IDI-1 and IDI-2 are “sluggish” enzymes with turnover times of ∼10 s–1 and ∼1 s–1, respectively. We measured incorporation of deuterium into IPP and DMAPP in D2O buffer for IDI-1 and IDI-2 under conditions where newly synthesized DMAPP is immediately and irreversibly removed by coupling its release to condensation with l-tryptophan catalyzed by dimethylallyltrytophan synthase. During the course of the reactions, we detected formation of d1, d2, and d3 isotopologues of IPP and DMAPP, which were formed during up to five isomerizations between IPP and DMAPP during each turnover. The patterns for deuterium incorporation into IPP ...
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kinetic and binding studies of streptococcus pneumoniae type 2 Isopentenyl Diphosphate dimethylallyl Diphosphate isomerase
Biochemistry, 2016Co-Authors: Matthew Walter Janczak, Dale C PoulterAbstract:Type 2 Isopentenyl Diphosphate:dimethylallyl Diphosphate isomerase (IDI-2) converts Isopentenyl Diphosphate (IPP) to dimethylallyl Diphosphate (DMAPP), the two fundamental building blocks of isoprenoid molecules. IDI-2 is found in many species of bacteria and is a potential antibacterial target since this isoform is non-homologous to the type 1 enzyme in Homo sapiens. IDI-2 requires a reduced flavin mononucleotide to form the catalytically active ternary complex, IDI-2·FMNH2·IPP. For IDI-2 from the pathogenic bacterium Streptococcus pneumoniae, the flavin can be treated kinetically as a dissociable cosubstrate in incubations with IPP and excess NADH. Under these conditions, the enzyme follows a modified sequential ordered mechanism where FMN adds before IPP. Interestingly, the enzyme shows sigmoidal behavior when incubated with IPP and NADH with varied concentrations of FMN in aerobic conditions. In contrast, sigmoidal behavior is not seen in incubations under anaerobic conditions where FMN is reduced to ...
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determination of kinetics and the crystal structure of a novel type 2 Isopentenyl Diphosphate dimethylallyl Diphosphate isomerase from streptococcus pneumoniae
ChemBioChem, 2014Co-Authors: Jerome De Ruyck, Rita M Cornish, Syam Sundar Neti, Steven Cary Rothman, Johan Wouters, Matthew Walter Janczak, Heidi L Schubert, Andre Matagne, Dale C PoulterAbstract:Isopentenyl Diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI-2) is a flavoenzyme found in bacteria that is completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady-state kinetic studies of the enzyme indicated that FMNH2 (KM =0.3 μM) bound before Isopentenyl Diphosphate (KM =40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 A resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence-similar and structure-related pathogens such as Enterococcus faecalis or Staphylococcus aureus.
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type 2 Isopentenyl Diphosphate isomerase evidence for a stepwise mechanism
Journal of the American Chemical Society, 2011Co-Authors: Nicole A Heaps, Dale C PoulterAbstract:Isopentenyl Diphosphate isomerase (IDI) catalyzes the interconversion of Isopentenyl Diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP). These two molecules are the building blocks for construction of isoprenoid carbon skeletons in nature. Two structurally unrelated forms of IDI are known. A variety of studies support a proton addition/proton elimination mechanism for both enzymes. During studies with Thermus thermophilus IDI-2, we discovered that the olefinic hydrogens of a vinyl thiomethyl analogue of Isopentenyl Diphosphate exchanged with solvent when the enzyme was incubated with D2O without concomitant isomerization of the double bond. These results suggest that the enzyme-catalyzed isomerization reaction is not concerted.
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type ii Isopentenyl Diphosphate isomerase probing the mechanism with alkyne allene Diphosphate substrate analogues
Biochemistry, 2010Co-Authors: Nagendra K Sharma, Jianjung Pan, Dale C PoulterAbstract: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 IDIs 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 or 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.
Hungwen Liu - One of the best experts on this subject based on the ideXlab platform.
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stereochemical studies of the type ii Isopentenyl Diphosphate dimethylallyl Diphosphate isomerase implicate the fmn coenzyme in substrate protonation
ChemBioChem, 2012Co-Authors: Jordi Calveras, Christopher J Thibodeaux, Steven O Mansoorabadi, Hungwen LiuAbstract:The interconversion of Isopentenyl Diphosphate (IPP, 1) and dimethylallyl Diphosphate (DMAPP, 2) by Isopentenyl Diphosphate:dimethylallyl Diphosphate isomerase (IDI) is a key reaction in the synthesis of the building blocks for isoprenoid compounds.[1] Two structurally unrelated types of IDI have been identified. The type I enzyme (IDI-1) is a zinc metalloprotein that also requires Mg2+ for activity.[2] It employs appropriate active site amino acid residues as general acid and base catalysts to carry out the isomerization reaction.[2g, 3] In contrast, the type II IDI (IDI-2), discovered in 2001,[4] is a flavoprotein that requires a reduced flavin mononucleotide (FMNred, 3) coenzyme in addition to Mg2+ for activity.[5] The reaction catalyzed by IDI-2 is unusual in that it utilizes the redox active FMN coenzyme to perform a reaction that does not involve a change in the redox state of the substrate/product. This observation raises questions as to the exact role of the flavin coenzyme in the catalytic mechanism. In addition, several human pathogens, such as Staphylococcus aureus, rely exclusively on IDI-2 for the initiation of long-chain isoprenoid biosynthesis, whereas humans employ the structurally unrelated IDI-1. Thus, IDI-2 is a potential target for new antimicrobial agents.
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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
Biochemistry, 2008Co-Authors: Christopher J Thibodeaux, Steven O Mansoorabadi, William Kittleman, Weichen Chang, Hungwen LiuAbstract:The type II Isopentenyl Diphosphate/dimethylallyl Diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of Isopentenyl pyrophos...
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characterization and mechanistic studies of type ii Isopentenyl Diphosphate dimethylallyl Diphosphate isomerase from staphylococcus aureus
Biochemistry, 2007Co-Authors: William Kittleman, Christopher J Thibodeaux, Hua Zhang, Yungnan Liu, Hungwen LiuAbstract: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.
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stereochemical analysis of Isopentenyl Diphosphate isomerase type ii from staphylococcus aureus using chemically synthesized s and r 2 2h Isopentenyl Diphosphates
Organic Letters, 2005Co-Authors: Chailin Kao, Haruo Seto, William Kittleman, Hua Zhang, Hungwen LiuAbstract: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.
C. D. Poulter - One of the best experts on this subject based on the ideXlab platform.
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covalent modification of reduced flavin mononucleotide in type 2 Isopentenyl Diphosphate isomerase by active site directed inhibitors
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Taku Nagai, C. D. Poulter, Hideaki Unno, Tohru Yoshimura, Matthew Walter Janczak, Hisashi HemmiAbstract: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 mechanism-based 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.
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covalent modification of reduced flavin mononucleotide in type 2 Isopentenyl Diphosphate isomerase by active site directed inhibitors
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Taku Nagai, C. D. Poulter, Hideaki Unno, Tohru Yoshimura, Matthew Walter Janczak, Hisashi HemmiAbstract: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 mechanism-based 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.
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synthesis of r 2 2h Isopentenyl Diphosphate and determination of its enantiopurity by 2h nmr spectroscopy in a lyotropic medium
Organic Letters, 1999Co-Authors: Aquiles E Leyes, C. D. PoulterAbstract:The synthesis of (R)-[2-2H]Isopentenyl Diphosphate from d-mannitol 1,2:5,6-bis-acetonide in 10 steps is reported. Stereospecific incorporation of the label is achieved by a BF3-catalyzed NaCNBD3 reduction of the enantiomerically pure (S)-isopropylidene oxirane intermediate. The enantiomeric excess of the penultimate precursor [2-2H]Isopentenyl tosylate (>95% ee) was determined by 2H NMR spectroscopy in a poly-γ-benzyl-l-glutamate/CH2Cl2 liquid crystal at −50 °C.
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analysis of the Isopentenyl Diphosphate isomerase gene family from arabidopsis thaliana
Plant Molecular Biology, 1998Co-Authors: Michael A Campbell, C. D. Poulter, F M Hahn, Thomas LeustekAbstract:Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding Isopentenyl Diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.
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Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for Isopentenyl Diphosphate isomerase.
Journal of bacteriology, 1996Co-Authors: Frederick M. Hahn, Jonathan A. Baker, C. D. PoulterAbstract:Isopentenyl Diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl Diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.
Steven Cary Rothman - One of the best experts on this subject based on the ideXlab platform.
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determination of kinetics and the crystal structure of a novel type 2 Isopentenyl Diphosphate dimethylallyl Diphosphate isomerase from streptococcus pneumoniae
ChemBioChem, 2014Co-Authors: Jerome De Ruyck, Rita M Cornish, Syam Sundar Neti, Steven Cary Rothman, Johan Wouters, Matthew Walter Janczak, Heidi L Schubert, Andre Matagne, Dale C PoulterAbstract:Isopentenyl Diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI-2) is a flavoenzyme found in bacteria that is completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady-state kinetic studies of the enzyme indicated that FMNH2 (KM =0.3 μM) bound before Isopentenyl Diphosphate (KM =40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 A resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence-similar and structure-related pathogens such as Enterococcus faecalis or Staphylococcus aureus.
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crystal structure of type 2 Isopentenyl Diphosphate isomerase from thermus thermophilus in complex with inorganic pyrophosphate
Biochemistry, 2008Co-Authors: Jerome De Ruyck, Dale C Poulter, Steven Cary Rothman, Jenny Pouyez, Johan WoutersAbstract:The N-terminal region is stabilized in the crystal structure of Thermus thermophilus type 2 Isopentenyl Diphosphate isomerase in complex with inorganic pyrophosphate, providing new insights about the active site and the catalytic mechanism of the enzyme. The PP i moiety is located near the conserved residues, H10, R97, H152, Q157, E158, and W219, and the flavin cofactor. The putative active site of Isopentenyl Diphosphate isomerase 2 provides interactions for stabilizing a carbocationic intermediate similar to those that stabilize the intermediate in the well-established protonation-deprotonation mechanism of Isopentenyl Diphosphate isomerase 1.
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type ii Isopentenyl Diphosphate isomerase irreversible inactivation by covalent modification of flavin
Journal of the American Chemical Society, 2008Co-Authors: Steven Cary Rothman, Joel R Walker, Jonathan B Johnston, Sungwon Lee, Dale C PoulterAbstract:Isopentenyl Diphosphate isomerase (IDI) catalyzes the interconversion of Isopentenyl Diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP), the basic 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; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl Diphosphate (eIPP), 3-methylene-4-penten-1-yl Diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl Diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV−visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-...
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synthesis and evaluation of substrate analogues as mechanism based inhibitors of type ii Isopentenyl Diphosphate isomerase
Journal of Organic Chemistry, 2008Co-Authors: Joel R Walker, Steven Cary Rothman, Dale C PoulterAbstract:Type 2 Isopentenyl Diphosphate isomerase (IDI-2), which catalyzes the interconversion of Isopentenyl Diphosphate and dimethylallyl Diphosphate, contains a tightly bound molecule of FMN. To probe the mechanism of the reaction, cyclopropyl and epoxy substrate analogues, designed to be mechanism-based irreversible inhibitors, were synthesized and evaluated with IDI-2 from Thermus thermophilus. The cyclopropyl analogues were alternative substrates. The epoxy analogue was an irreversible inhibitor, with kI = 0.37 ± 0.07 min-1 and KI = 1.4 ± 0.3 μM. LC−MS studies revealed formation of an epoxide-FMN adduct.
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type 2 Isopentenyl Diphosphate isomerase mechanistic studies with cyclopropyl and epoxy analogues
Journal of the American Chemical Society, 2007Co-Authors: Jonathan B Johnston, Joel R Walker, Steven Cary Rothman, Dale C PoulterAbstract:Type-2 Isopentenyl Diphosphate isomerase, which catalyzes the interconversion of Isopentenyl Diphosphate and dimethylallyl Diphosphate, contains a tightly bound molecule of FMN. Incubation of the a...
Hisashi Hemmi - One of the best experts on this subject based on the ideXlab platform.
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substrate induced change in the quaternary structure of type 2 Isopentenyl Diphosphate isomerase from sulfolobus shibatae
Journal of Bacteriology, 2012Co-Authors: Hitomi Nakatani, Shuichiro Goda, Hideaki Unno, Takuya Nagai, Tohru Yoshimura, Hisashi HemmiAbstract:Type 2 Isopentenyl Diphosphate isomerase catalyzes the interconversion between two active units for isoprenoid biosynthesis, i.e., Isopentenyl Diphosphate and dimethylallyl Diphosphate, in almost all archaea and in some bacteria, including human pathogens. The enzyme is a good target for discovery of antibiotics because it is essential for the organisms that use only the mevalonate pathway to produce the active isoprene units and because humans possess a nonhomologous isozyme, type 1 Isopentenyl Diphosphate isomerase. However, type 2 enzymes were reportedly inhibited by mechanism-based drugs for the type 1 enzyme due to their surprisingly similar reaction mechanisms. Thus, a different approach is now required to develop new inhibitors specific to the type 2 enzyme. X-ray crystallography and gel filtration chromatography revealed that the enzyme from a thermoacidophilic archaeon, Sulfolobus shibatae, is in the octameric state at a high concentration. Interestingly, a part of the regions that are involved in the substrate binding in the previously reported tetrameric structures is integral to the formation of the tetramer-tetramer interface in the substrate-free octameric structure. Site-directed mutagenesis at such regions resulted in stabilization of the tetramer. Small-angle X-ray scattering, tryptophan fluorescence, and dynamic light scattering analyses showed that substrate binding causes the dissociation of an octamer into tetramers. This property, i.e., incompatibility between octamer formation and substrate binding, might provide clues to develop new specific inhibitors of the archaeal enzyme.
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covalent modification of reduced flavin mononucleotide in type 2 Isopentenyl Diphosphate isomerase by active site directed inhibitors
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Taku Nagai, C. D. Poulter, Hideaki Unno, Tohru Yoshimura, Matthew Walter Janczak, Hisashi HemmiAbstract: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 mechanism-based 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.
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covalent modification of reduced flavin mononucleotide in type 2 Isopentenyl Diphosphate isomerase by active site directed inhibitors
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Taku Nagai, C. D. Poulter, Hideaki Unno, Tohru Yoshimura, Matthew Walter Janczak, Hisashi HemmiAbstract: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 mechanism-based 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.
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new role of flavin as a general acid base catalyst with no redox function in type 2 Isopentenyl Diphosphate isomerase
Journal of Biological Chemistry, 2009Co-Authors: Hideaki Unno, Tokuzo Nishino, Satoshi Yamashita, Toru Nakayama, Yosuke Ikeda, Tohru Yoshimura, Shin Ya Sekiguchi, Norie Yoshida, Masami Kusunoki, Hisashi HemmiAbstract:Using FMN and a reducing agent such as NAD(P)H, type 2 Isopentenyl-Diphosphate isomerase catalyzes isomerization between Isopentenyl Diphosphate and dimethylallyl Diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 Isopentenyl-Diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.
