Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclizations of geranyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.     

Biosynthesis of monoterpenes: demonstration of a geranyl pyrophosphate:(-)-bornyl pyrophosphate cyclase in soluble enzyme preparations from tansy (Tanacetum vulgare)

Tansy (Tanacetum vulgare L.) produces an essential oil containing the optically pure monoterpene ketone, (-)-camphor, as a major constituent. A soluble enzyme preparation from immature leaves of this plant converts the acyclic precursor [1-3H]geranyl pyrophosphate to the bicyclic monoterpene alcohol borneol in the presence of MgCl2, and oxidizes a portion of the borneol to camphor in the presence of a pyridine nucleotide. The identity of the major biosynthetic product as borneol was confirmed by chemical oxidation to camphor and crystallization of the derived oxime to constant specific radioactivity. The stereochemistry of the borneol was verified as the (-)-(1S,4S) isomer by oxidation to camphor, conversion to the corresponding ketal with D-(-)-2,3-butanediol, and separation of diastereoisomers by radio-gas-liquid chromatography. When enzyme reaction mixtures were treated with a mixture of acid phosphatase and apyrase, following an initial ether extraction of labeled borneol, additional quantities of borneol were generated, indicating the presence of a phosphorylated derivative of borneol. This water-soluble metabolite was prepared by large-scale enzyme incubations with [1-3H]geranyl pyrophosphate (plus phosphatase inhibitor), and the identity of the initial cyclization product was established as (-)-bornyl pyrophosphate by direct ion-exchange chromatographic analysis and enzymatic hydrolysis. The pathway for the formation of (-)-(1S,4S)-camphor was therefore identical to that previously demonstrated for the (+)-(1R,4R) isomer, involving cyclization of geranyl pyrophosphate to bornyl pyrophosphate, hydrolysis of this intermediate to borneol, and oxidation of the alcohol to the ketone. The labeling pattern of the product derived from [1-3H2, U-14C]geranyl pyrophosphate was determined by oxidation of the biosynthetic borneol to camphor and selective removal of tritium by exchange of the alpha hydrogens at C3 of the ketone. This labeling pattern was identical to that observed previously for the (+) isomer, suggesting the same mechanism of cyclization, but of opposite enantiospecificity. Some properties of the antipodal (+)- and (-)-bornyl pyrophosphate cyclases were compared.     

Metabolism of monoterpenes: demonstration that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint (Mentha piperita)

Piperitenone is commonly considered to be the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint; however, [3H]piperitenone gave rise only to the inert metabolite (+)-piperitone when incubated with peppermint leaf discs. Under identical conditions, (-)-[3H]isopiperitenone was efficiently incorporated into (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and yielded an additional metabolite identified as (+)-cis-isopulegone; piperitenone was poorly labeled. Moreover, (+)-cis-[3H]isopulegone was rapidly converted to (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and the reduction of (+)-[3H]pulegone to (-)-menthone and (+)-isomenthone was similarly documented. Each step of the pathway was demonstrated in a crude soluble preparation from peppermint leaf epidermis and each of the relevant enzymes was partially purified in order to compare relative rates of catalysis. The results of these studies indicate that the endocyclic double bond of (-)-isopiperitenone is reduced to yield (+)-cis-isopulegone, which is isomerized to (+)-pulegone as the immediate precursor of (-)-menthone and (+)-isomenthone, and they rule out piperitenone as an intermediate of the pathway.     

Biosynthesis of monoterpenes. Stereochemical implications of acyclic and monocyclic olefin formation by (+)- and (-)-pinene cyclases from sage

(+)-Pinene cyclase from sage (Salvia officinalis) catalyzes the isomerization and cyclization of geranyl pyrophosphate to (+)-alpha-pinene and (+)-camphene, and to lesser amounts of (+)-limonene, myrcene, and terpinolene, whereas (-)-pinene cyclase from this tissue catalyzes the conversion of the acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene, and to lesser quantities of (-)-limonene, myrcene, and terpinolene. The bicyclic products of these enzymes (pinene and camphene) are derived via the cyclization of the cisoid, anti-endo-conformers of the bound, tertiary allylic intermediates (3R)-linalyl pyrophosphate [+)-pinene cyclase) and (3S)-linalyl pyrophosphate [-)-pinene cyclase). When challenged with either enantiomer of linalyl pyrophosphate or with neryl pyrophosphate (cis-isomer of geranyl pyrophosphate) as substrate, both pinene cyclases synthesize disproportionately high levels of acyclic olefins (myrcene and ocimene) and monocyclic olefins (limonene and terpinolene), compared with the product mixtures generated from the natural geranyl precursor. Resolution of the limonene derived from linalyl pyrophosphate and neryl pyrophosphate demonstrated that this monocyclic olefin was formed via conformational foldings in addition to the cisoid,anti-endo-pattern. These results indicate that the alternate substrates are ionized by the cyclases prior to their achieving the optimum orientation for bicyclization. In the case of geranyl pyrophosphate, a preassociation mechanism is suggested in which optimum folding of the terpenyl chain precedes the initial ionization step.     

(-)-Linalool inhibits in vitro NO formation: Probable involvement in the antinociceptive activity of this monoterpene compound

Recent studies performed in our laboratory have shown that (-)-linalool, the natural occurring enantiomer in essential oils, possesses anti-inflammatory, antihyperalgesic and antinociceptive effects in different animal models. The antinociceptive and antihyperalgesic effect of (-)-linalool has been ascribed to the stimulation of the cholinergic, opioidergic and dopaminergic systems, to its local anaesthetic activity and to the blockade of N-Methyl-d-aspartate receptors (NMDA). Since nitric oxide (NO) and prostaglandin E(2) (PGE(2)) play an important role in oedema formation and hyperalgesia and nociception development, to investigate the mechanism of these actions of the (-)-linalool, we examined the effects of this compound on lipopolysaccharide (LPS)-induced responses in macrophage cell line J774.A1. Exposure of LPS-stimulated cells to (-)-linalool significantly inhibited nitrite accumulation in the culture medium without inhibiting the LPS-stimulated increase of inducible nitric oxide synthase (iNOS) expression, suggesting that the inhibitory activity of (-)-linalool is mainly due to the iNOS enzyme activity. In contrast, exposure of LPS-stimulated cells to (-)-linalool failed, if not at the highest concentration, both in inhibiting PGE(2) release and in inhibiting increase of inducible cyclooxygenase-2 (COX(2)) expression in the culture medium. Collectively, these results indicate that the reduction of NO production/release is responsible, at least partially, for the molecular mechanisms of (-)-linalool antinociceptive effect, probably through mechanisms where cholinergic and glutamatergic systems are involved.     

Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita).

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor,saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.     

Site-directed mutagenesis to probe protein folding: evidence that the formation and aggregation of a bovine growth hormone folding intermediate are dissociable processes

Bovine growth hormone (bGH) forms a stable folding intermediate that aggregates at elevated concentrations (greater than 10 microM). Thermodynamic and kinetic studies have shown that the formation of this bGH folding intermediate and its aggregation are separate processes, implying that selective modifications of bGH can lead to their independent modulation. In addition, a bGH region that includes amino acid residues 109-133 appears to be directly involved in this aggregation process. Human growth hormone (hGH), which is unable to aggregate via this mechanism, differs from the bovine primary sequence at eight positions within this protein region. We have characterized the folding of a bGH analogue that contains the hGH sequence between amino acid residues 109-133 (8H-bGH) at low and high concentrations. The equilibrium folding characteristics of bGH and 8H-bGH are similar when monitored at low protein concentrations (less than or equal to 2 microM). The wild-type and analogue proteins have equivalent denaturation midpoints when equilibrium unfolding is monitored by the use of far-UV circular dichroism, second-derivative UV, or fluorescence. In addition, the enhanced fluorescence that is associated with the formation of the bGH monomeric folding intermediate (Havel, H. A., et al. (1988) Biochim. Biophys. Acta 955, 154-163) is observed for 8H-bGH under similar conditions. In contrast, partial denaturation of 8H-bGH at higher concentrations (greater than 2 microM) leads to significantly less aggregation than is observed for bGH. This result is obtained from near-UV CD spectroscopy, kinetic folding, size-exclusion chromatography, and dynamic light-scattering data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita) and spearmint (Mentha spicata).

The p-menthane monoterpenes of the Mentha species are biosynthesized from geranyl pyrophosphate via the monocyclic olefin 4S-limonene. A monoterpene cyclase was isolated from both Mentha x piperita (peppermint) and Mentha spicata (spearmint) that catalyzes the cyclization of geranyl pyrophosphate to 4S-limonene. This enzyme, 4S-limonene synthase, was purified to apparent homogeneity by dye ligand, anion exchange, and hydrophobic interaction chromatography. Since the monoterpenes of Mentha are synthesized and secreted in modified epidermal hairs called glandular trichomes, an extract of isolated glandular trichome cells was used as the source of this enzyme. A combination of gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that purified 4S-limonene synthase had a native molecular weight of 56,000 and was monomeric. The principal product of the enzyme was enantiomerically pure (-)-4S-limonene, and a catalytic constant of 0.3/s was determined. The basic properties of 4S-limonene synthase from both M. x piperita and M. spicata are identical and, in general, are similar to those of other monoterpene, sesquiterpene, and diterpene cyclases isolated from microorganisms and higher plants.     

Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor-bean endosperm.

Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.     

Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes that are hydrolyzed by paraoxonase (PON-1) during high density lipoprotein oxidation with a peroxynitrite donor

High density lipoprotein (HDL) is rich in polyunsaturated phospholipids that are sensitive to oxidation. However, the effect of apolipoprotein A-I and paraoxonase-1 (PON-1) on phosphatidylcholine oxidation products has not been identified. We subjected native HDL, trypsinized HDL, and HDL lipid suspensions to oxidation by the peroxynitrite donor, 3-morpholinosydnonimine. HDL had a basal level of phosphatidylcholine mono- and di-hydroperoxides that increased to a greater extent in HDL, compared with either trypsinized HDL or HDL lipid alone. Phosphatidylcholine core aldehydes, which were present in small amounts, increased 10-fold during oxidation of native HDL, compared with trypsinized HDL (p = 0.004), and 4-fold compared with HDL lipid suspensions (p = 0.0021). In addition, the content of lysophosphatidylcholine increased 300% during oxidation of native HDL, but only 80 and 25%, respectively, during oxidation of trypsinized HDL and HDL lipid suspensions. Phosphatidylcholine isoprostanes accumulated in comparable amounts during the oxidation of all three preparations. Incubation of apolipoprotein A-I with 1-palmitoyl-2-linoleoyl glycerophosphocholine proteoliposomes in the presence of 3-morpholinosydnonimine or apoAI with phosphatidylcholine hydroperoxides resulted in a significant increase in phosphatidylcholine core aldehydes with no formation of lysophosphatidylcholine. We propose that apolipoprotein A-I catalyzes a one-electron oxidation of alkoxyl radicals. Purified PON-1 hydrolyzed phosphatidylcholine core aldehydes to lysophosphatidylcholine. We conclude that, upon HDL oxidation with peroxynitrite, apolipoprotein AI increases the formation of phosphatidylcholine core aldehydes that are subsequently hydrolyzed by PON1.     

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.     

Collapsin response mediator proteins (CRMPs) are a new class of microtubule-associated protein (MAP) that selectively interacts with assembled microtubules via a taxol-sensitive binding interaction

Collapsin response mediator proteins are ubiquitously expressed from multiple genes (CRMPs 1-5) and play important roles in dividing cells and during semaphorin 3A (Sema3A) signaling. Nonetheless, their mode of action remains opaque. Here we carried out in vivo and in vitro assays that demonstrate that CRMPs are a new class of microtubule-associated protein (MAP). In experiments with CRMP1 or CRMP2 and their derivatives, only the C-terminal region (residues 490-572) mediated microtubule binding. The in vivo microtubule association of CRMPs was abolished by taxol or epothilone B, which is highly unusual. CRMP2-depleted cells exhibited destabilized anaphase astral microtubules and altered spindle position. In a cell-based assay, all CRMPs stabilized interphase microtubules against nocodazole-mediated depolymerization, with CRMP1 being the most potent. Remarkably, a 82-residue C-terminal region of CRMP1 or CRMP2, unrelated to other microtubule binding motifs, is sufficient to stabilize microtubules. In cells, we demonstrate that glycogen synthase kinase-3β (GSK3β) inhibition potentiates this activity. Thus, CRMPs are a new class of MAP that binds through a unique motif, but in common with others such as Tau, is antagonized by GSK3β. This regulation is consistent with such kinases being critical for the Sema3A (collapsin) pathway. These findings have implications for cancer and neurodegeneration.     

Biosynthesis of monoterpenes: Preliminary characterization of l-endo-fenchol synthetase from fennel (Foeniculum vulgare) and evidence that no free intermediate is involved in the cyclization of geranyl pyrophosphate to the rearranged product

A soluble enzyme preparation from the leaves of fennel (Foeniculum vulgare M.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpene l-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980 Arch. Biochem. Biophys.200, 524–533). To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography on O-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled out endo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene, cis-2-pinanol, trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. The endo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn²⁺ (1 mm) for catalytic activity. Co²⁺ can partially substitute for Mn²⁺, but other divalent cations are ineffective. The partially purified synthetase is inhibited by p-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase.     

Observation of myo-inositol 1,2-(cyclic) phosphate in a Morris hepatoma by 31P NMR

We have identified an unusual resonance at 16.5 ppm in the 31P NMR spectrum of a Morris (7777) hepatoma grown in the inguinal fossa of a Buffalo rat as myoinositol 1,2-(cyclic) phosphate. This compound has been observed in all of the 32 tumors examined as well as in cultured cells derived from the tumor, but it has not been observed in normal rat tissues. Its level in the aqueous phase of chloroform/methanol/water extracts of the tumor is 70 +/- 40 nmol/g, wet weight (n = 4). The presence of a breakdown product of phosphatidylinositol at such high levels in a fast growing tumor may provide an important clue for understanding the metabolic defect that results in the malignant growth of this tumor.     

A novel method of producing the pharmaceutical intermediate (R)-2-chloromandelic acid by bioconversion

(R)-2-Chloromandelic acid (R­CM) is one of the chiral building blocks used in the pharmaceutical industry. As a result of screening for microorganisms that asymmetrically hydrolyze racemic 2­chloromandelic acid methyl ester (CMM), Exophiala dermatitidis NBRC6857 was found to produce R­CM at optical purity of 97% ee. The esterase that produces R­CM, EstE, was purified from E. dermatitidis NBRC6857, and the optimal temperature and pH of EstE were 30°C and 7.0, respectively. The estE gene that encodes EstE was isolated and overexpressed in Escherichia coli JM109. The activity of recombinant E. coli JM109 cells overexpressing estE was 553 times higher than that of E. dermatitidis NBRC6857. R­CM was produced at conversion rate of 49% and at optical purity of 97% ee from 10% CMM with 0.45 mg-dry-cell/L recombinant E. coli JM109 cells. Based on these findings, R­CM production by bioconversion of CMM may be of interest for future industrial applications.     

Evidence that the effects of arginine-8-vasopressin (AVP) on pituitary corticotropin (ACTH) release are mediated by a novel type of receptor

Ovine corticotropin releasing factor (oCRF-41) and AVP act synergistically to stimulate pituitary ACTH secretion. In the present study we have investigated whether the effect of AVP, either in the presence or in the absence of oCRF-41 (0.5 nmol/l), could be blocked by V1 (pressor)-antagonists. Furthermore, oxytocin, and [1-deamino,8-D-arginine] vasopressin (dDAVP) were tested for their ability to release ACTH. All experiments were carried out in vitro, using segments of rat anterior pituitary glands. The V1-antagonist [1-deamino,penicillamine(o-methyl-tyrosine)]AVP inhibited ACTH release induced by AVP or AVP + oCRF-41. However, it also had some agonistic activity which was more pronounced in the presence of oCRF-41. An equally potent V1-antagonist, [1-beta-mercapto-beta, beta-cyclopentamethyleneproprionic acid (o-methyl-tyrosine)]AVP, failed to inhibit AVP-stimulated ACTH secretion, and also had weak agonist potency. The relatively selective V2 (antidiuretic)-agonist dDAVP was 20-30 fold less potent than AVP. Oxytocin, a weak V1- and V2-agonist was only 4-8 fold less potent than AVP. These data are compatible with the suggestion that AVP receptors on pituitary corticotrope cells are neither classical V1- nor V2-receptors.     

Mechanism of monoterpene cyclization: stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (-)-limonene synthase from spearmint and (-)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated analog and homolog of the tertiary intermediate linalyl diphosphate (i.e., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively). In the case of recombinant (+)-bornyl diphosphate synthase from common sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization-cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction.