Catalytic Mechanism of Escherichia coli Isopentenyl Diphosphate Isomerase Involves Cys-67, Glu-116, and Tyr-104 as Suggested by Crystal Structures of Complexes with Transition State Analogues and Irreversible Inhibitors

Wouters, Johan; Oudjama, Yamina; Barkley, Sam J.; Tricot, Catherine; Stalon, Victor; Droogmans, Louis; Poulter, C. Dale

doi:10.1074/jbc.m212823200

Cited by 73 publications

(83 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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)(3)(4)(5). In contrast, IDI-2 is a flavoprotein that requires reduced FMN for activity (6,7).…”

mentioning

confidence: 83%

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.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 83%

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.

Self Cite

View full text Add to dashboard Cite

show abstract

“…It is possible that its catalytic function is most similar to that of the type II isopentenyl diphosphate (IPP)/ dimethylallyl diphosphate (DMAPP) isomerase, which converts IPP to DMAPP by isomerizing a double bond (62,63). Both type I and type II IPP/DMAPP isomerases appear to catalyze isomerization by stereoselective protonation and deprotonation of the substrate (64,65), and the type II IPP isomerase, like CrtY, requires a reduced flavin and NADPH for activity (62,63,64). In this case, although there is no net oxidation or reduction, both reduction and oxidation of the substrate occur during the reaction, and the flavin may have a role in these half-reactions.…”

Section: Discussionmentioning

confidence: 99%

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

Maresca

Graham

et al. 2007

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

133

View full text Add to dashboard Cite

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL-or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA-and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL-and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.carotenogenesis ͉ carotenoids ͉ cyanobacteria ͉ green sulfur bacteria ͉ photosynthesis C olored carotenoids are found in nearly all photosynthetic species and in a variety of nonphotosynthetic organisms and play roles in light-harvesting, photoprotection, structural maintenance of pigment-protein complexes (1), and membrane structure and fluidity (2). The cyclization of the linear compound lycopene to produce ␣-, ␤-, ␥-, or -carotene is a branch point in carotenoid biosynthetic pathways in many species of bacteria, plants, and fungi (3, 4). The monocyclic ␥-carotene is a precursor to myxoxanthophylls in cyanobacteria (5, 6), is both an intermediate and an end product of carotenogenesis in orange and yellow flowers and fruits (7-10), and is an intermediate in chlorobactene biosynthesis in green sulfur bacteria (GSB) (11-13). Dicyclic ␤-carotene is a major component of photosystems (PS) I and II in cyanobacteria and plants (14,15) and is modified to isorenieratene in brown-colored GSB and some actinomycetes (16,17).Three classes of lycopene cyclases have previously been identified in bacteria: the CrtY-type ␤-cyclases that are found in many carotenogenic proteobacteria (e.g., refs. 18 and 19), Streptomyces spp. (17), and the Chloroflexi; the CrtL family, which includes the ␤-and -cyclases in some cyanobacteria and plants (20,21); and the heterodimeric cyclases of some Gram-positive bacteria (22, 23), which are related to the lycopene cyclases of archaea (24, 25) and halophilic bacteria (26). These classes are distantly related to each other and share only a few conserved motifs, including an N-terminal flavin-binding domain that is found in the first two groups but appears to be missing in the third (27). Carotenoid monocyclases are found in both the CrtY and CrtL families (28-30), and there are no obvious sequence differences between mono-and dicyclases (28).The cyclization of lycopene to ␥-or ␤-carotene is an isomerization reaction, which produces no net change in mass or redox state o...

show abstract

“…Complex was obtained by soaking crystals of the Y104F mutant with 3,4-epoxy-3-methyl-1-butyl diphosphate (EIPP), a mechanism-based irreversible inhibitor (8). Solution of the inhibitor (25 mM) in Tris maleate (100 mM, pH 4.5), PEG 2000 mme (14%), ammonium sulfate (100 mM), MnCl 2 and MgCl 2 (20 mM), and glycerol (25%) was replaced every 3 h for at least 24 h.…”

Section: Methodsmentioning

confidence: 99%

“…However, these studies have not yet conclusively established the origin of the proton activating the double bond in IPP. Glu-116 is likely the critical acidic residue that drives protonation by lowering the energy of the carbocation as demonstrated by the crystallographic structure of the transition-state analogue N,N-dimethyl-2-amino-1-ethyl diphosphate in complex with the enzyme (8). However, Glu-116 is probably not protonated because it is directly coordinated to the catalytic metal cation.…”

mentioning

confidence: 99%

“…Crystal structures of free and metal-bound Escherichia coli IDI-1 show that the first divalent cation is involved in the active conformation folding, with the metal occupying a first coordination site composed of three histidines and two glutamates (7). The structures of E. coli IPP isomerase in complex with diphosphate substrate analogues (N,N-dimethyl-2-amino-1-ethyl diphosphate (8), 3,4-epoxy-3-methyl-1-butyl diphosphate (8,9), and the bromohydrin of isopentenyl diphosphate (10)) show the presence of the second metal site occupied by Mg 2ϩ cation. Carbonyl oxygen of residue 67, side chain oxygen of Glu-87, two water molecules, and two nonbridging oxygens of the diphosphate moiety of the inhibitors coordinate this metal.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Structural Role for Tyr-104 in Escherichia coli Isopentenyl-diphosphate Isomerase

Ruyck

Durisotti²,

Oudjama³

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase.Isoprenoids 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 isoprenoid compounds are characterized by the basic isoprene unit. Nature uses two activated isoprene units to build these prenoid compounds as follows: isopentenyl diphosphate (IPP) 3 and its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP) (Scheme 1). Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase (IDI; EC 5.3.3.2) is a key enzyme involved in the biosynthesis of isoprenoids and catalyzes the isomerization of IPP into DMAPP (2). DMAPP then condenses with additional molecules of IPP to form farnesyl diphosphate, which is required for protein prenylation, cholesterol biosynthesis, and synthesis of a variety of higher molecular weight isoprenoids. Although the synthesis of IPP can occur through the mevalonate pathway in which acetyl-CoA and mevalonate are substrates, a mevalonate-independent pathway producing IPP and DMAPP was found in plant chloroplasts, algae, cyanobacteria, and some bacteria. This quite new pathway, known as the deoxyxylulose 5-phosphate pathway, begins with pyruvate and glyceraldehyde 3-phosphate as the initial substrates.Two types of IDI are reported. They show no sequence similarity but catalyze the same reaction. Type I enzyme is widely distributed in microorganisms. Type II IPP isomerase is a flavoprotein that belongs to the FMN-dependent ␣-hydroxyacid dehydrogenase family and displays a classical TIM barrel fold (3, 4). Type I IPP isomerase is composed of about 200 amino acids and folds into a compact globular protein that belongs to the class of ␣/␤ proteins. It is a metalloprotein containing divalent cations, Zn 2ϩ and Mg 2ϩ , ascofactors (5, 6). Crystal structures of free and metal-bound Escherichia coli IDI-1 show that the first divalent cation is involved in the active conformation folding, with the metal occupying a first coordination site composed of three histidines and two glutamates (7). The structures of E. coli IPP isomerase in complex with diphosphate substrate analogues (N,N-dimethyl-2-amino-1-ethyl diphosphate (8), 3,4-epoxy-3-methyl-1-butyl diphosphate (8, 9), and the b...

show abstract

Catalytic Mechanism of Escherichia coli Isopentenyl Diphosphate Isomerase Involves Cys-67, Glu-116, and Tyr-104 as Suggested by Crystal Structures of Complexes with Transition State Analogues and Irreversible Inhibitors

Cited by 73 publications

References 20 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

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

Structural Role for Tyr-104 in Escherichia coli Isopentenyl-diphosphate Isomerase

Contact Info

Product

Resources

About