Crystal Structure of Cucumene Synthase, a Terpenoid Cyclase That Generates a Linear Triquinane Sesquiterpene.

Linear triquinanes are sesquiterpene natural products with hydrocarbon skeletons consisting of three fused five-membered rings. Importantly, several of these compounds exhibit useful anticancer, anti-inflammatory, and antibiotic properties. However, linear triquinanes pose significant challenges to organic synthesis because of the structural and stereochemical complexity of their hydrocarbon skeletons. To illuminate nature's solution to the generation of linear triquinanes, we now describe the crystal structure of Streptomyces clavuligerus cucumene synthase. This sesquiterpene cyclase catalyzes the stereospecific cyclization of farnesyl diphosphate to form a linear triquinane product, (5 S,7 S,10 R,11 S)-cucumene. Specifically, we report the structure of the wild-type enzyme at 3.05 Å resolution and the structure of the T181N variant at 1.96 Å resolution, both in the open active site conformations without any bound ligands. The high-resolution structure of T181N cucumene synthase enables inspection of the active site contour, which adopts a three-dimensional shape complementary to a linear triquinane. Several aromatic residues outline the active site contour and are believed to facilitate cation-π interactions that would stabilize carbocation intermediates in catalysis. Thus, aromatic residues in the active site not only define the template for catalysis but also play a role in reducing activation barriers in the multistep cyclization cascade.

Mechanistic Studies of the Protonation-Deprotonation Reactions for Type 1 and Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase.

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 show that d2-IPP is formed in preference to d1-IPP for both enzymes. Analysis of the patterns of deuterium incorporation are consistent with a mechanism involving addition and removal of protons by a concerted asynchronous process, where addition substantially precedes removal, or a stepwise process through a short-lived (<3 ps) tertiary carbocationic intermediate. Previous work with mechanism-based inhibitors and related model studies supports a concerted asynchronous mechanism for the enzyme-catalyzed isomerizations.

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus.

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

Structure-Function Studies of Artemisia tridentata Farnesyl Diphosphate Synthase and Chrysanthemyl Diphosphate Synthase by Site-Directed Mutagenesis and Morphogenesis.

The amino acid sequences of farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the "regular" sesquiterpenoid farnesyl diphosphate (FPP) by coupling isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and then to geranyl diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the "irregular" monoterpenes chrysanthemyl diphosphate (CPP), lavandulyl diphosphate (LPP), and trace quantities of maconelliyl diphosphate (MPP) from two molecules of DMAPP, and couples IPP to DMAPP to give GPP. A. tridentata FPPase and CPPase belong to the chain elongation protein family (PF00348), a subgroup of the terpenoid synthase superfamily (CL0613) whose members have a characteristic α terpene synthase α-helical fold. The active sites of A. tridentata FPPase and CPPase are located within a six-helix bundle containing amino acids 53 to 241. The two enzymes were metamorphosed into one another by sequentially replacing the loops and helices of the six-helix bundle from enzyme with those from the other. Chain elongation was the dominant activity during the N-terminal to C-terminal metamorphosis of FPPase to CPPase, with product selectivity gradually switching from FPP to GPP, until replacement of the final α-helix, whereupon cyclopropanation and branching activity competed with chain elongation. During the corresponding metamorphosis of CPPase to FPPase, cyclopropanation and branching activities were lost upon replacement of the first helix in the six-helix bundle. Mutations of active site residues in CPPase to the corresponding amino acids in FPPase enhanced chain-elongation activity, while similar mutations in the active site of FPPase failed to significantly promote formation of significant amounts of irregular monoterpenes. Our results indicate that CPPase, a promiscuous enzyme, is more plastic toward acquiring new activities, whereas FPPase is more resistant. Mutations of residues outside of the α terpene synthase fold are important for acquisition of FPPase activity for synthesis of CPP, LPP, and MPP.

Further Insight into Crystal Structures of Escherichia coli IspH/LytB in Complex with Two Potent Inhibitors of the MEP Pathway: A Starting Point for Rational Design of New Antimicrobials.

IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7 Šresolution, respectively. The structure of the E. coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.

Defining the Product Chemical Space of Monoterpenoid Synthases.

Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes.

Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase.

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 FMNH2 before the reaction is initiated by addition of IPP. Stopped-flow experiments revealed that FMN, whether bound to IDI-2 or without enzyme in solution, is slowly reduced in a pseudo-first-order reaction upon addition of excess NADH (k(red)(FMN) = 5.7 × 10(-3) s(-1) and k(red)(IDI-2·FMN) = 2.8 × 10(-3) s(-1)), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2·FMN, and IPP (k(red)(IDI-2·FMN·IPP) = 8.9 s(-1)). Similar experiments with dithionite as the reductant gave k(red)(FMN) = 221 s(-1) and k(red)(IDI-2·FMN) = 411 s(-1). Dithionite reduction of FMN in the IDI-2·FMN and IPP mixture was biphasic with k(red)(IDI-2·FMN·IPP (fast)) = 326 s(-1) and k(red)(IDI-2·FMN·IPP (slow)) = 6.9 s(-1) The pseudo-first-order rate constant for the slow component was similar to those for NADH reduction of the flavin in the IDI-2·FMN and IPP mixture and may reflect a rate-limiting conformational change in the enzyme.

Construction of Functional Monomeric Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase.

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the isoprenoid biosynthetic pathway. The enzyme from Streptomyces pneumoniae (spIDI-2) is a homotetramer in solution with behavior, including a substantial increase in the rate of FMN reduction by NADPH in the presence of IPP, suggesting that substrate binding at one subunit alters the kinetic and binding properties of another. We now report the construction of catalytically active monomeric spIDI-2. The monomeric enzyme contains a single-point mutation (N37A) and a six-residue C-terminal deletion that preserves the secondary structure of the subunits in the wild-type (wt) homotetramer. UV-vis spectra of the enzyme-bound flavin mononucleotide (FMN) cofactor in FMNox, FMNred, and FMNred·IPP/DMAPP states are the same for monomeric and wt homotetrameric spIDI-2. The mutations in monomeric IDI-2 lower the melting temperature of the protein by 20 °C and reduce the binding affinities of FMN and IDI by 40-fold but have a minimal effect on kcat. Stopped-flow kinetic studies of monomeric spIDI-2 showed that the rate of reduction of FMN by NADH (k = 1.64 × 10(-3) s(-1)) is substantially faster when IPP is added to the monomeric enzyme (k = 0.57 s(-1)), similar to behavior seen for wt-spIDI-2. Our results indicate that cooperative interactions among subunits in the wt homotetramer are not responsible for the increased rate of reduction of spIDI-2·FMN by NADH, and two possible scenarios for the enhancement are suggested.

Site-Selective Synthesis of (15)N- and (13)C-Enriched Flavin Mononucleotide Coenzyme Isotopologues.

Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with (15)N and (13)C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis.

Synthesis and Enzymatic Studies of Isoprenoid Thiolo Bisubstrate Analogues.

Chain elongation prenyltransferases catalyze the addition of the hydrocarbon moiety of allylic isoprenoid diphosphates to the carbon-carbon double bond in isopentenyl diphosphate (IPP) in the primary building reactions in the isoprenoid biosynthetic pathway. Bis-O-diphosphate analogues 3-OPP/OPP, 4-OPP/OPP, and 5-OPP/OPP and bis-thiolodiphosphate bisubstrate analogues 3-SPP/SPP, 4-SPP/SPP, and 5-SPP/SPP were synthesized. The analogues 4-OPP/OPP, 5-OPP/OPP, 4-SPP/SPP, and 5-SPP/SPP were excellent competitive inhibitors of avian farnesyl diphosphate synthase with KI = 1.0 ± 0.12 µM, KI = 0.5 ± 0.2 µM, KI = 0.7 ± 0.3 µM, and KI = 2.9 ± 0.27 µM, respectively, whereas, analogues 3-OPP/OPP and 3-SPP/SPP displayed mixed type inhibition with KI = 1.4 µM and KI = 5.5 µM, respectively.

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily.

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme-ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e(-70)) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.

Predicting the functions and specificity of triterpenoid synthases: a mechanism-based multi-intermediate docking approach.

Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed.

Synthesis and enzymatic studies of bisubstrate analogues for farnesyl diphosphate synthase.

Farnesyl diphosphate synthase catalyzes the sequential chain elongation reactions between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP) and between IPP and GPP to give farnesyl diphosphate (FPP). Bisubstrate analogues containing the allylic and homoallylic substrates were synthesized by joining fragments for IPP and the allylic diphosphates with a C-C bond between the methyl group at C3 in IPP and the Z-methyl group at C3 in DMAPP (3-OPP) and GPP (4-OPP), respectively. These constructs placed substantial limits on the conformational space available to the analogues relative to the two substrates. The key features of the synthesis of bisubstrate analogues 3-OPP and 4-OPP are a regioselective C-alkylation of the dianion of 3-methyl-3-buten-1-ol (5), a Z-selective cuprate addition of alkyl groups to an α,ß-alkynyl ester intermediate, and differential activation of allylic and homoallylic alcohols in the analogues, followed by a simultaneous displacement of the leaving groups with tris(tetra-n-butylammonium) hydrogen diphosphate to give the corresponding bisdiphosphate analogues. The bisubstrate analogues were substrates for FPP synthase, giving novel seven-membered ring analogues of GPP and FPP. The catalytic efficiencies for cyclization of 3-OPP and 4-OPP were similar to those for chain elongation with IPP and DMAPP.

Isoprenoid Biosynthesis in Pathogenic Bacteria: Nuclear Resonance Vibrational Spectroscopy Provides Insight into the Unusual [4Fe-4S] Cluster of the E.†coli LytB/IspH Protein.

The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate-free LytB has a special [4Fe-4S](2+) cluster with a yet unsolved structure. This motivated us to use synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical-molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate-free LytB. The apical iron atom of the [4Fe-4S](2+) is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) and to the inhibitors (E)-4-amino-3-methylbut-2-en-1-yl diphosphate and (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate.

Determination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae.

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 Å 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.

Regio- and chemoselective immobilization of proteins on gold surfaces.

Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by ß-mercaptoethanol than alkane-thiol SAMs.

Sandwich antibody arrays using recombinant antibody-binding protein L.

Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a "sandwich-type" antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months.

δ-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates.

The biosynthetic pathways to isoprenoid compounds involve transfer of the prenyl moiety in allylic diphosphates to electron-rich (nucleophilic) acceptors. The acceptors can be many types of nucleophiles, while the allylic diphosphates only differ in the number of isoprene units and stereochemistry of the double bonds in the hydrocarbon moieties. Because of the wide range of nucleophilicities of naturally occurring acceptors, the mechanism for prenyltransfer reactions may be dissociative or associative with early to late transition states. We have measured δ-secondary kinetic isotope effects operating through four bonds for substitution reactions with dimethylallyl derivatives bearing deuterated methyl groups at the distal (C3) carbon atom in the double bond under dissociative and associative conditions. Computational studies with density functional theory indicate that the magnitudes of the isotope effects correlate with the extent of bond formation between the allylic moiety and the electron-rich acceptor in the transition state for alkylation and provide insights into the structures of the transition states for associative and dissociative alkylation reactions.

Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.

The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes.

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

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.