Cloning and functional characterization of an isopentenyl diphosphate isomerase gene from Tripterygium wilfordii.

Tripterygium wilfordii Hook.F. is one of the most valuable medicinal plants because it contains a large variety of active terpenoid compounds, including triptolide, celastrol, and wilforlide. All of the pharmacologically active secondary metabolites are synthesized from the 2-C-methyl-d-erythritol 4-phosphate and mevalonate pathway in the isoprenoid biosynthetic system. The key step in this pathway is the isomerization of dimethylallyl diphosphate and isopentenyl diphosphate, which is catalyzed by isopentenyl diphosphate isomerase (IPI). In the present study, a full-length cDNA encoding IPI (designate as TwIPI, GenBank accession no.KT279355) was cloned from a suspension of cultured cells from T. wilfordii. The full-length cDNA of TwIPI was 1,564 bp and encoded a polypeptide of 288 amino acids. The bioinformatics analysis showed that the deduced TwIPI sequence contained the TNTCCSHPL and WGEHELDY motif. The transcription level of the TwIPI in the suspension cells increased almost fivefold after treatment with methyl jasmonate as an elicitor. A functional color assay in Escherichia coli indicated that TwIPI could promote the accumulation of lycopene and encoded a functional protein.

Structure of mycobacterial ß-oxidation trifunctional enzyme reveals its altered assembly and putative substrate channeling pathway.

The incidence of tuberculosis is increasing due to the appearance of new drug-resistant variants. A thorough understanding of the disease organism is essential in order to create more effective drugs. In an attempt to understand better the poorly studied lipid metabolism of Mycobacterium tuberculosis (Mtb), we identified and characterized its fatty acid ß-oxidation complex (trifunctional enzyme (TFE)). TFE is an α(2)ß(2) complex consisting of two types of polypeptides catalyzing three of the four reactions of the ß-oxidation of fatty acids. The kinetic constants (k(cat) and K(m)) show that the complexed α chain is more active than the individual α chain. Crystal structures of Mtb TFE (mtTFE) reveal that the quaternary assembly is strikingly different from the already known Pseudomonas fragi TFE (pfTFE) assembly due to the presence of a helical insertion (LA5) in the mtTFE-ß subunit. This helical insertion prevents the pfTFE mode of assembly, as it would clash with helix H9A of the TFE-α chain. The mtTFE assembly appears to be more rigid and results in a different substrate channeling path between the α and the ß subunits. Structural comparisons suggest that the mtTFE active sites can accommodate bulkier fatty acyl chains than in pfTFE. Although another thiolase (FadA2), more closely related to human TFE-ß/thiolase, is present in the Mtb genome, it does not form a complex with mtTFE-α. Extensive phylogenetic analyses show that there are at least four TFE subfamilies. Our studies highlight the molecular properties of mtTFE, significantly extending the structural knowledge on this type of very interesting multifunctional enzymes.

Cloning, expression and characterization of lepidopteran isopentenyl diphosphate isomerase.

Isopentenyl diphosphate isomerase (IPPI) of the spruce budworm, Choristoneura fumiferana, and of the tobacco hornworm, Manduca sexta, was cloned and its catalytic properties assessed. In the presence of Mg(2+) or Mn(2+), the recombinant protein from C. fumiferana (CfIPPI) efficiently isomerized IPP to dimethylallyl diphosphate (DMAPP). While C. fumiferana IPPI transcript levels were evenly distributed in a wide variety of tissues, they were highly abundant in the corpora allata. Because IPPI plays an alternate role in lepidopteran juvenile hormone (JH) biosynthesis by catalyzing the isomerization of the homologous substrate, homoisopentenyl diphosphate (HIPP), the ability of CfIPPI to convert HIPP to homodimethylallyl diphosphate (HDMAPP) was also studied. As expected, HIPP isomerization was efficient and the formation of HDMAPP occurred, but the regiospecificity of the reaction was lower than previously found in M. sexta corpora allata homogenates and with purified Bombyx mori IPPI. Differences in inhibitory potency for several alkylated ammonium diphosphates and higher homologs of DMAPP were noted between CfIPPI and a vertebrate IPPI, suggesting that the lepidopteran enzyme has a larger active site cavity. To determine the structural factors responsible for homologous substrate coupling, site directed mutagenesis of several residues identified through sequence alignment and homology modeling analysis was performed. The results suggest that unlike other IPPIs, W216 (C. fumiferana numbering) works in concert with a tyrosine residue (Y105) to allow binding of larger substrates and to stabilize the high-energy intermediate formed during substrate isomerization.

Crystal structure and putative mechanism of 3-methylitaconate-delta-isomerase from Eubacterium barkeri.

3-Methylitaconate-Delta-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K(m) but > 1000-fold lower k(cat). The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 A. The asymmetric unit of the P2(1)2(1)2(1) unit cell with parameters a = 53.1 A, b = 142.3 A, and c = 228.4 A contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an alpha/beta-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Delta-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.

A new isopentenyl diphosphate isomerase gene from Camptotheca acuminata: cloning, characterization and functional expression in Escherichia coli.

Isopentenyl diphosphate isomerase (EC 5.3.3.2, IPI) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which are the essential precursors for isoprenoids, including anti-tumor camptothecin. Here we report cloning, characterization and functional expression of a new cDNA encoding IPI from Camptotheca acuminata. The full-length cDNA was 1143 bp long designated as CaIPI (GenBank Accession Number: DQ839416), containing an open reading frame (ORF) of 930bp which encodes a polypeptide of 309 amino acids. Bioinformatic analysis showed the cDNA sequence of CaIPI was highly homologous with other IPI gene and the deduced amino acid sequence of CaIPI was similar to known plant IPIs and contained Cys-149 and Glu-212 active sites. Phylogenic analysis indicated that all IPIs could be divided into five groups and CaIPI belonged to plant IPIs' family. The tissue expression profile analysis was carried out to investigate the transcriptional level of CaIPI in different tissues. The result showed that CaIPI expression could be detected in roots, stems and tender leaves but could not in mature leaves and fruits, and the expression levels was much higher in stems than in roots and tender leaves. Finally, CaIPI was functionally expressed in engineered Escherichia coli in which the carotenoid pathway was reconstructed. In engineered E. coli, CaIPI could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orange beta-carotene. These confirmed that CaIPI had the typically function of IPI gene. In summary, cloning, characterization and functional expression of CaIPI will facilitate to understand the function of CaIPI at the level of molecular genetics and unveil the biosynthetic mechanism of camptothecin precursors.

Structure and mechanism of the Propionibacterium acnes polyunsaturated fatty acid isomerase.

Conjugated linoleic acids (CLAs) affect body fat gain, carcinogenesis, insulin resistance, and lipid peroxidation in mammals. Several isomers of CLA exist, of which the (9Z, 11E) and (10E, 12Z) isomers have beneficial effects on human metabolism but are scarce in foods. Bacterial polyunsaturated fatty acid isomerases are promising biotechnological catalysts for CLA production. We describe six crystal structures of the Propionibacterium acnes polyunsaturated fatty acid isomerase PAI in apo- and product-bound forms. The three-domain flavoprotein has previously undescribed folds outside the FAD-binding site. Conformational changes in a hydrophobic channel toward the active site reveal a unique gating mechanism for substrate specificity. The geometry of the substrate-binding site explains the length preferences for C18 fatty acids. A catalytic mechanism for double-bond isomerization is formulated that may be altered to change substrate specificity for syntheses of rare CLAs from easily accessible precursors.

Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase.

Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.

Structural basis for channelling mechanism of a fatty acid beta-oxidation multienzyme complex.

The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid beta-oxidation cycle. The alpha2beta2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3'-phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3'-phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The alpha-helical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other beta-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.

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.

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 A) of complexes of Escherichia coli IPP.DMAPPs 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.

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.

Characterization of PECI, a novel monofunctional Delta(3), Delta(2)-enoyl-CoA isomerase of mammalian peroxisomes.

We report here the identification and characterization of human and mouse PECI, a novel gene that encodes a monofunctional peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase. Human and mouse PECI were identified on the basis of their sequence similarity to Eci1p, a recently characterized peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae. Cloning and sequencing of the human PECI cDNA revealed the presence of a 1077-base pair open reading frame predicted to encode a 359-amino acid protein with a mass of 39.6 kDa. The corresponding mouse cDNA contains a 1074-base pair open reading frame that encodes a 358-amino acid-long protein with a deduced mass of 39.4 kDa. Northern blot analysis demonstrated human PECI mRNA is expressed in all tissues. A bacterially expressed form of human PECI catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 27 units/mg of protein. The human and mouse PECI proteins contain type-1 peroxisomal targeting signals, and human PECI was localized to peroxisomes by both subcellular fractionation and immunofluorescence microscopy techniques. The potential roles for this monofunctional Delta(3),Delta(2)-enoyl-CoA isomerase in peroxisomal metabolism are discussed.

Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte.

The enzyme isopentenyl pyrophosphate (IPP) isomerase catalyzes the reversible isomerization of IPP to produce dimethylallyl pyrophosphate, the initial substrate leading to the biosynthesis of carotenoids and many other long-chain isoprenoids. Expression of IPP isomerase, and of two enzymes specific to the carotenoid pathway (lycopene beta-cyclase and beta-carotene-C-4-oxygenase), was followed in the green unicellular alga Haematococcus pluvialis after exposure to high illumination. This alga uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of ketocarotenoids in the cytosol. The carotenoid/chlorophyll ratio increased 3-fold in wild type and 6-fold in a precocious carotenoid-accumulating mutant (Car-3) within 24 h after increasing the illumination from 20 to 150 micromol photon m-2.s-1. Two cDNAs encoding IPP isomerase in Haematococcus, ipiHp1 and ipiHp2, were identified. Although otherwise highly similar (95% identity overall), the predicted sequence of ipiHp1 contained a 12-aa region not found in that of ipiHp2. This was reflected by a size difference between two polypeptides of 34 and 32.5 kDa, both of which reacted with an antibody to the product of ipiHp1. We suggest that the 32.5-kDa form is involved with the carotenoid accumulation in the cytoplasm, since the 32.5-kDa polypeptide was preferentially up-regulated by high light preceding the carotenoid increase and only this form was detected in red cysts.

Delta3,5-delta2,4-dienoyl-CoA isomerase from rat liver. Molecular characterization.

rECH1, a recently identified rat cDNA (FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Genomics 27, 457-466) encodes a polypeptide belonging to the hydratase/isomerase superfamily. We modeled the structure of rECH1 based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts that rECH1p has the hydratase fold in the core domain and two domains for interaction with other subunits. When we incubated 3,5,8,11, 14-eicosapentaenoyl-CoA with purified rECH1p, the spectral data suggested a switching of the double bonds from the Delta3-Delta5 to the Delta2-Delta4 positions. This was confirmed by demonstrating that the product was a valid substrate for 2,4-dienoyl-CoA reductase. These results indicate that rECH1p is Delta3,5-Delta2,4-dienoyl-CoA isomerase. Subcellular fractionation and immunoelectron microscopy using antibodies to a synthetic polypeptide derived from the C terminus of rECH1p showed that rECH1p is located in the matrix of both mitochondria and peroxisomes in rat liver. Consistent with these observations, the 36,000-Da rECH1p has a potential N-terminal mitochondrial targeting signal as well as a C-terminal peroxisomal targeting signal type 1. Transport of the protein into the mitochondria with cleavage of the targeting signal results in a mature mitochondrial form with a molecular mass of 32,000 Da; transport to peroxisomes yields a protein of 36,000 Da.