Structure of limonene synthase, a simple model for terpenoid cyclase catalysis

The crystal structure of (4S)-limonene synthase from Mentha spic ata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-A; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate-metal ion coordination and substrate binding conformation in the course of the multistep reaction.     

Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site

We used in silico methods to screen a library of 1,013 compounds for possible binding to the allosteric site in farnesyl diphosphate synthase (FPPS). Two of the 50 predicted hits had activity against either human FPPS (HsFPPS) or Trypanosoma brucei FPPS (TbFPPS), the most active being the quinone methide celastrol (IC₅₀ versus TbFPPS ∼20 µM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC₅₀ values in the range of ∼1–3 µM (as compared with ∼0.5 µM for the bisphosphonate inhibitor, zoledronate). The three leads were the quinone methides taxodone and taxodione and the quinone arenarone, compounds with known antibacterial and/or antitumor activity. We then obtained X-ray crystal structures of HsFPPS with taxodione+zoledronate, arenarone+zoledronate, and taxodione alone. In the zoledronate-containing structures, taxodione and arenarone bound solely to the homoallylic (isopentenyl diphosphate, IPP) site, not to the allosteric site, whereas zoledronate bound via Mg²⁺ to the same site as seen in other bisphosphonate-containing structures. In the taxodione-alone structure, one taxodione bound to the same site as seen in the taxodione+zoledronate structure, but the second located to a more surface-exposed site. In differential scanning calorimetry experiments, taxodione and arenarone broadened the native-to-unfolded thermal transition (T_m), quite different to the large increases in ΔT_m seen with biphosphonate inhibitors. The results identify new classes of FPPS inhibitors, diterpenoids and sesquiterpenoids, that bind to the IPP site and may be of interest as anticancer and antiinfective drug leads.Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of isopentenyl diphosphate (IPP; compound 1 in Fig. 1) with dimethylallyl diphosphate (DMAPP; compound 2 in Fig. 1) to form the C₁₀ isoprenoid geranyl diphosphate (GPP; compound 3 in Fig. 1), which then condenses with a second IPP to form the C₁₅ isoprenoid, farnesyl diphosphate (FPP; compound 4 in Fig. 1). FPP then is used in a wide range of reactions including the formation of geranylgeranyl diphosphate (GGPP) (1), squalene (involved in cholesterol and ergosterol biosynthesis), dehydrosqualene (used in formation of the Staphylococcus aureus virulence factor staphyloxanthin) (2), undecaprenyl diphosphate (used in bacterial cell wall biosynthesis), and quinone and in heme a/o biosynthesis. FPP and GGPP also are used in protein (e.g., Ras, Rho, Rac) prenylation, and FPPS is an important target for the bisphosphonate class of drugs (used to treat bone resorption diseases) such as zoledronate (compound 5 in Fig. 1) (3). Bisphosphonates targeting FPPS have activity as antiparasitics (4), act as immunomodulators (activating γδ T cells containing the Vγ2Vδ2 T-cell receptor) (5), and switch macrophages from an M2 (tumor-promoting) to an M1 (tumor-killing) phenotype (6). They also kill tumor cells (7) and inhibit angiogenesis (8). However, the bisphosphonates in clinical use (zoledronate, alendronate, risedronate, ibandronate, etidronate, and clodronate) are very hydrophilic and bind avidly to bone mineral (9). Therefore, there is interest in developing less hydrophilic species (10) that might have better activity against tumors in soft tissues and better antibacterial (11) and antiparasitic activity.Open in a separate windowFig. 1.Chemical structures of FPPS substrates, products, and inhibitors.The structure of FPPS (from chickens) was first reported by Tarshis et al. (12) and revealed a highly α-helical fold. The structures of bacterial and Homo sapiens FPPS (HsFPPS) are very similar; HsFPPS structure (13, 14) is shown in Fig. 2A. There are two substrate-binding sites, called here “S1” and “S2.” S1 is the allylic (DMAPP, GPP) binding site to which bisphosphonates such as zoledronate bind via a [Mg²⁺]₃ cluster (15) (Fig. 2B). S2 is the homoallylic site to which IPP binds, Fig. 2B. Recently, Jahnke et al. (10) and Salcius et al. (16) discovered a third ligand-binding site called the “allosteric site” (hereafter the “A site”). A representative zoledronate+A-site inhibitor structure [Protein Data Bank (PDB) ID code 3N46] (Nov_980; compound 6 in Fig. 1) showing zoledronate in S1 and Nov_980 (compound 6) in the A site is shown in a stereo close-up view in Fig. 2B, superimposed on a zoledronate+IPP structure (PDB ID code 2F8Z) in S2. Whether the allosteric site serves a biological function (e.g., in feedback regulation) has not been reported. Nevertheless, highly potent inhibitors (IC₅₀ ∼80 nM) have been developed (10), and the best of these newly developed inhibitors are far more hydrophobic than are typical bisphosphonates (∼2.4–3.3 for cLogP vs. ∼−3.3 for zoledronate) and are expected to have better direct antitumor effects in soft tissues (10).Open in a separate windowFig. 2.Structures of human FPPS. (A) Structure of HsFPPS showing zoledronate (compound 5) and IPP (compound 1) bound to the S1 (allylic) and S2 (homoallylic) ligand-binding sites (PDB ID code 2F8Z). (B) Superposition of the IPP-zoledronate structure (PDB ID code 2F8Z) on the zoledronate-Nov_980 A-site inhibitor structure (PDB ID code 3N46). Zoledronate binds to the allylic site S1, IPP binds to the homoallylic site S2, and the allosteric site inhibitor binds to the A site. Active-site “DDXXD” residues are indicated, as are Mg²⁺ molecules (green and yellow spheres, respectively). The views are in stereo.In our group we also have developed more lipophilic compounds (e.g., compound 7 in Fig. 1) (17, 18) as antiparasitic (19) and anticancer drug leads (18) and, using computational methods, have discovered other novel nonbisphosphonate FPPS inhibitors (e.g., compound 8 in Fig. 1) that have micromolar activity against FPPS (20). In this study, we extended our computational work and tried to discover other FPPS inhibitors that target the A site. Such compounds would be of interest because they might potentiate the effects of zoledronate and other bisphosphonates, as reported for other FPPS inhibitors (21), and have better tissue distribution properties in general.     

Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement.

Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterpene precursor geranylgeranyl diphosphate to the stable bicyclic intermediate copalyl diphosphate. In the second, magnesium ion-dependent reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone and is coupled, by intramolecular proton transfer within a transient pimarenyl intermediate, to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane structure. Alternative deprotonations of the terminal abietenyl carbocation provide a mixture of abietadiene, levopimaradiene, and neoabietadiene, and this product profile varies as a function of pH. Mutational analysis of amino acids at the active site of a modeled structure has identified residues critical for catalysis, as well as several that play roles in specifying product formation, apparently by ligation of a magnesium ion cofactor. These results strongly suggest that choice between alternatives for deprotonation of the abietenyl intermediate depends more on the positioning effects of the carbocation-diphosphate anion reaction partners than on the pKa of multiple participating bases. In one extreme case, mutant N765A is unable to mediate the intramolecular proton transfer and aborts the reaction, without catalyzing 1,2-methyl migration, to produce only sandaracopimaradiene, thereby providing supporting evidence for the corresponding stereochemistry of the cryptic pimarenyl intermediate of the reaction pathway.     

Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer

Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer.     

Cloning of casbene synthase cDNA: evidence for conserved structural features among terpenoid cyclases in plants.

A near-full-length casbene synthase cDNA clone, pCS7, was isolated by using a partial cDNA clone, pCS4, to probe a lambda gt10 library constructed from poly(A)+ RNA from elicited castor bean seedlings. The cDNA insert had a length of 1983 bases with a polyadenylate tail of 19 bases. Translation of the cDNA sequence revealed an open reading frame encoding a 601-aa protein with a predicted M(r) of 68,960. Search of the GenBank data base with the deduced translation product revealed 42% identity and 65% similarity with 5-epi-aristolochene synthase from tobacco and 31% identity and 53% similarity with limonene synthase from spearmint. Each of the three proteins catalyzes an intramolecular cyclization of a prenyl diphosphate substrate to a specific cyclic terpenoid hydrocarbon product. The proposed reaction mechanisms for the three catalytic processes share common chemical features, even though the products being formed are members of three different classes of terpenoid compounds. Analysis of the alignment of the three proteins suggests that both primary and secondary structural elements are conserved. These similarities suggest that the genes that encode terpenoid cyclization enzymes of this type in angiosperms have undergone divergent evolution from an ancestral progenitor gene. In support of this proposition, the locations of five of the six introns in the casbene synthase gene align very closely with those of the five introns in the 5-epi-aristolochene synthase gene.     

Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.     

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.     

Primary structure and functional expression of a glutaminyl cyclase.

A glutaminyl cyclase (QC) that is probably involved in the biosynthesis of pyroglutamyl peptides such as gonadotropin-releasing hormone and thyrotropin-releasing hormone has been purified to homogeneity from bovine anterior pituitary. On the basis of N-terminal sequence analysis, a 2088-base-pair cDNA clone was isolated from a bovine anterior pituitary library. From the nucleotide sequence of this clone, the primary structure of a 330-residue protein and a preceding 31-residue prepropeptide sequence was deduced. By transfection of COS-7 monkey cells with a QC cDNA/pCDM8 vector construct, QC activity was expressed. Hybridization with mRNAs of various bovine tissues revealed expression of QC mainly in brain tissue.     

Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase.

The intestinal hormone guanylin and bacterial heat-stable enterotoxins (STs) are members of a peptide family that activates intestinal membrane guanylate cyclase. Two different peptides that activate the human intestinal T84 cell guanylate cyclase have been purified from urine and intestinal mucosa of opossums (Didelphis virginiana). The highly acidic peptide, QEDCELCINVACTGC, was named uroguanylin because it was isolated from urine and shares 53% identity with guanylin. A second peptide, SHTCEICAFAACAGC, was purified from urine and intestinal mucosa. This alanine-rich peptide was 47% identical to uroguanylin and 73% identical to human guanylin, suggesting that it may be an opossum homologue of guanylin. Synthetic uroguanylin-(2-15) (i.e., EDCELCINVACTGC) was 10-fold more potent than synthetic rat guanylin, but both peptides were less potent than Escherichia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 125I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepithelial Cl- secretion was stimulated by 1 microM uroguanylin, indicated by an increase in the short circuit current of T84 cells. Thus, uroguanylin is another paracrine hormone in the emerging peptide family that activates intestinal membrane guanylate cyclase. The second peptide may be the opossum form of guanylin, or perhaps, it is still another member of this peptide family. The presence of uroguanylin and guanylin in urine and receptors in proximal tubules suggests that these peptides may also originate from renal tissue and may regulate kidney function.     

Altered glucagon- and catecholamine hormone-sensitive adenylyl cyclase responsiveness in rat liver membranes induced by manipulation of dietary fatty acid intake

Glucagon-stimulated adenylyl cyclase activity has been shown to change in liver membranes manipulated to alter either their fatty acid composition or fluidity. We examined whether membrane alterations induced by dietary manipulation affected receptor function. Glucagon- and beta-adrenergic-stimulated receptor-adenylyl cyclase systems were examined in liver membranes of rats fed diets containing 10% corn oil, 10% coconut oil (essential FFA deficient), or 8.5% coconut oil with 1.5% corn oil (essential FFA repleat). Basal and maximal nonreceptor-mediated adenylyl cyclase activity (stimulated by NaF, guanylylimidodiphosphate, and forskolin) was the same in membranes of each of the dietary groups, suggesting that Gs-protein and the catalytic unit activity per se were unaltered by the manipulations. Glucagon-stimulated adenylyl cyclase activity increased with increasing unsaturation of dietary fatty acids; activity in coconut oil-fed rats was 527 +/- 30 (mean +/- SEM) pmol/mg.10 min, that in coconut/corn oil-fed rats was 752 +/- 74 pmol/mg.10 min, and that in corn oil-fed rats was 981 +/- 94 pmol cAMP/mg.10 min. [125I]Monoiodoglucagon binding did not increase in parallel to the adenylyl cyclase alterations; coconut oil-fed animals (614 fmol/mg) differed from the other groups (450 and 430 fmol/mg). Isoproterenol (beta-adrenergic)-stimulated adenylyl cyclase activity was also highest in the corn oil-fed animals, but was similar in the other dietary groups, with no difference in other characteristics of [125I]iodopindolol binding between the groups. The results demonstrate that alterations in the glucagon-stimulated adenylyl cyclase response are different from those in the beta-adrenergic adenylyl cyclase response. Further, they suggest that although direct activations of the catalytic unit or its interaction with the guanine nucleotide-sensitive protein are apparently not affected, hormone receptor-mediated adenylyl cyclase activity may be altered by these dietary manipulations.     

A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.     

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: a promising neuroprotective strategy.

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(S)-N-[4-[4-[(3,4-dihydro-6-hydroxy-2, 5,7, 8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2- thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties in vitro, affords remarkable neuronal protection in vivo. Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3-10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.     

Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.

Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-.     

Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control

A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits high-level levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/L was subsequently obtained by cultivation in a bench-scale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products.