Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state. Analysis of the crystal structures (2 Å) of complexes of Escherichia coliIPP⅐DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/ elimination of protons during isomerization. This work provides a new perspective about the mechanism of the reaction. Isopentenyl diphosphate (IPP)1 :dimethylallyl diphosphate (DMAPP) isomerase (EC 5.3.3.2) catalyzes the rearrangement of IPP (Scheme 1, 1) to its electrophilic allylic isomer DMAPP (2). This is a mandatory activation step of the isoprenoid unit for subsequent prenyl transfer reactions in isoprenoid biosynthesis. Isomerization of the carbon-carbon double bond in IPP involves an antarafacial (1, 3) transposition of hydrogen by a proton addition-elimination mechanism (1, 2). During the reaction, a proton is added to the re-face of the double bond in IPP, and the re-proton at C2 is removed (3).In eukaryotes, IPP is synthesized by the mevalonate pathway and is the exclusive product from phosphorylation and decarboxylation of mevalonic acid. IPP isomerase activity is an essential enzyme for these organisms. In many bacteria and in plant chloroplasts, IPP and DMAPP are both synthesized from 4-hydroxydimethylallyl diphosphate by the methylerythritol phosphate pathway (4). Although IPP isomerase is often found in these bacteria, it is not essential. Multiple sequence alignments indicate that Escherichia coli IPP isomerase is related to the eukaryotic enzymes (5). In addition, a combination of gas chromatography/mass spectrometry and NMR studies with labeled IPP showed that the stereochemistry for the reversible introduction and removal of a proton at C2 during interconversion of IPP and DMAPP is the same for E. coli IPP isomerase and the eukaryotic enzymes (6).Several lines of evidence support the protonation/deprotonation mechanism shown in Scheme 1. These include proton exchange measurements (7), decreased reactivity for a fluorinated analogue of DMAPP, potent noncovalent inhibition by ammonium analogues of the putative carbocationic intermediate (8, 9), and irreversible inhibition by mechanism-based inhibitors containing epoxide moieties (9 -11). Based on the antarafacial stereochemistry for protonation/deprotonation, it has been proposed that IPP isomerase has two active-site bases located on opposite faces of the allylic moiety defined by C2-C3-C4 in IPP. One of these bases is in the conjugate acid form and protonates the double bond. The other assists with the subsequent elimination of a proton (9). Reardon and Abeles (8) presented evidence for involvement of a thiol group during catalysis. The thiol group a...

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His 6 -tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni The isoprenoid biosynthetic pathway is ubiquitous across all three kingdoms of life. Over 36,000 natural products derived from the fundamental five-carbon isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) have now been identified (3). Many of these molecules have important biological functions, including glycoprotein synthesis (dolichols), inter-and intracellular signaling (prenylated proteins and steroidal hormones), membrane structure (steroids), electron carriers during redox reactions (ubiquinones), photoprotection (carotenoids), photosynthesis (chlorophyll), and defense against predators (sesquiterpenes and pyrethrins).There are two independent biosynthetic routes to IPP and DMAPP (Fig. 1). In the mevalonate (MVA) pathway, first discovered in the late 1950s (4, 6), IPP is synthesized from mevalonic acid by the consecutive action of mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), and mevalonate diphosphate decarboxylase (DPMVA decarboxylase). The isomerization of IPP to DMAPP is a mandatory step needed to create the electrophilic allylic diphosphates needed for subsequent prenyl transfer reactions. IPP isomerase is an essential enzyme in archaea, eukaryota, and some gram-positive eubacteria, where IPP is synthesized by the MVA pathway. More recently, it was discovered that IPP and DMAPP are synthesized from D-glyceraldehyde phosphate and pyruvate by the methyl erythritol phosphate (MEP) pathway found in many eubacteria, cyanobacteria, and plant chloroplasts (18,28). In the final step of the MEP pathway, the ispH gene product synthesizes IPP and DMAPP from 4-hydroxydimethylallyl diphosphate (Fig. 1) (26). In organisms that utilize the MEP pathway, idi is not essential or does not exist (10).In 1997, Methanothermobacter thermautotrophicus was the first archaeon to have its genome fully sequenced (34). At th...

Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His 6 -tagged protein was produced in Escherichia coli and purified by Ni 2؉ chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg 2؉for activity; K m IPP was 52 M, and k cat IPP was 0.23 s ؊1 .The isomerization of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) is an essential reaction in the mevalonate (MVA) pathway to isoprenoids (13) and, although not essential, probably serves to balance the IPP and DMAPP pools in the methylerythritol phosphate (MEP) route (9). Two types of IPP isomerase are known. The type I enzyme, found in Eucarya and some of the Bacteria, is well characterized (16). Type II IPP isomerase, found in Archaea and some of the Bacteria, was discovered much more recently (10), and only a few studies of the protein have been reported. Analysis of the genome of Synechocystis sp. strain PCC 6803 indicated that the cyanobacterium synthesizes isoprenoid compounds by the MEP route. Although it was reported elsewhere that the bacterium does not have detectable IPP isomerase activity under phototrophic growth conditions (6, 7), open reading frame (ORF) sll1556 encodes a protein that is substantially similar to other type II IPP isomerases. We now report that purified ORF sll1556 protein is an active type II IPP isomerase.Evidence that Synechocystis sp. strain PCC 6803 ORF sll1556 encodes a functional type II IPP isomerase was obtained by complementation studies with Salmonella enterica serovar Typhimurium strain RMC29 (Table 1) (2). The chromosomal copy of idi (IPP isomerase) in RMC29 was disrupted with a chloramphenicol (CAM) marker, and dxs (deoxyxylulose synthase) was disrupted with a minioperon that includes the yeast genes required for biosynthesis of IPP from MVA and an ampicillin (AMP) marker. Thus, RMC29 is viable when supplemented with methylerythritol (ME) but does not grow on MVA unless idi activity is restored. RMC29 was transformed with plasmid pJMSB02278-8 bearing a copy of ORF sll1556 from Synechocystis sp. strain PCC 6803 and the expression plasmid without ORF sll1556. Both strains were resistant to AMP and expressed the plasmid-carried genes when induced with arabinose. As shown in Fig. 1, strains JMSB02278-8, JMSB0354, and RMC29 grew on LB broth-CAM-ME, demonstrating that they can utilize the MEP pathway to synthesize IPP and DMAPP without a functional IPP isomerase. Strains JMSB02278-8 and JMSB0354 contain plasmids conferring resistance to AMP and grew on LB-AMP-CAM-ME, while RMC29 did not. Only JMSB02278-8 grew on LB-AMP-CAM-MEV-arabinose by utilizing the IPP isomerase encoded by ORF sll1556 from Synechocystis sp. strain PCC 6803 to convert IPP synthesized from MVA to DMAPP.Nickel-nitrilotriacetic acid affinity chromatography of the supernatant from a cell extract of Escherichia coli strain JMSB0373a yielded a protein that gave a single band upon sodium dodecyl sulfate-polyacrylami...

[reaction: see text] Type II isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase from Synechocystis PCC 6803 catalyzes the interconversion of IPP and DMAPP. Upon incubation of the enzyme with IPP or DMAPP in 2H2O, one deuterium is incorporated into the C2 methylene of IPP, two deuteriums are incorporated at C4, and three deuteriums are incorporated into the (E)-methyl of DMAPP.