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R Croteau

Publications and source records attributed to R Croteau.

At least 73 records · Page 4Linked to original sources
PubMed1996

Partial purification and characterization of a monoterpene cyclase, limonene synthase, from the liverwort Ricciocarpos natans.

4S-(-)-limonene is the major monoterpene accumulated by the liverwort Ricciocarpos natans, and a cell-free extract from this nonvascular plant cultured in vitro catalyzes the cyclization of geranyl diphosphate to limonene. The time course of limonene synthase activity parallels cultured growth but shows a maximum in specific activity in the lag phase following transfer to fresh medium. The operationally soluble enzyme was partially purified by combination of anion-exchange and hydroxylapatite chromatography. The limonene synthase from R. natans possesses a molecular weight of about 51,000, exhibits a pH optimum at 6.5, a pI at 5.3, and a requirement for either Mg2+ or Mn2+ as cofactor, and appears to employ 3S-linalyl disphosphate as a bound intermediate in the cyclization of the geranyl substrate (Km approximately 1.25 microM). In stereochemistry of the coupled isomerization-cyclization reaction and in general properties, the limonene synthase from this bryophyte resembles the corresponding monoterpene cyclases from gymnosperm and angiosperm species.

Cyclohexenes↗
PubMed1996

Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

Antibodies↗
PubMed1996

A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis.

The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa-4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton. To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases. Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems. Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced. One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa-4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry. The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir.

Amino Acid Sequence↗
PubMed1996

Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew.

The first committed step in the formation of taxol has been shown to involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer, taxa-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-2H2,20-2H3] and [20-2H3]geranylgeranyl diphosphates were employed as substrates with the partially purified taxadiene synthase from Pacific yew (Taxus brevifolia) stems to examine the possibility of a preliminary cyclization to taxa-4(20),11(12)-diene followed by isomerization to the more stable endocyclic double bond isomer. GLC-MS analysis of the derived taxa-4(5),11(12)-diene, via selected ion monitoring of the parent ion and the P-15 and C-ring fragment ions, compared to those of unlabeled standard, showed the olefin product to possess a deuterium enrichment essentially identical to that of the acyclic precursor, thus ruling out the putative isomerization step. With [4-2H2]geranylgeranyl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5),11(12)-diene to contain only one deuterium atom, consistent with direct formation from a taxenyl cation by deprotonation at C5. (+/-)-Casbene, (+/-)-verticillene, and (+/-)-taxa-4(20),11(12)-diene were tested as possible olefinic intermediates in taxa-4(5),11(12)-diene formation by a series of inhibition, trapping, and direct conversion experiments; no evidence was obtained that these exogenous olefins could serve as intermediates of the cyclization reaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-3H]geranylgeranyl diphosphate in 2H2O indicated little incorporation of deuterium from the medium and suggested a rapid internal proton transfer in a tightly bound olefinic intermediate. Analysis of the enzymatic product generated from [10-2H1]geranylgeranyl diphosphate confirmed the intramolecular hydrogen transfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5),11(12)-diene. From these results, a stereochemical mechanism is proposed for the taxadiene synthase reaction involving the initial cyclization of geranylgeranyl diphosphate to a transient verticillyl cation intermediate, with transfer of the C11 alpha-proton to C7 to initiate transannular B/C-ring closure to the taxenyl cation, followed by deprotonation at C5 to yield the taxa-4(5),11(12)-diene product directly.

Isomerases↗
PubMed1996

Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol: the first oxygenation step in taxol biosynthesis.

BACKGROUND: The structural complexity of taxol dictates continued reliance on biological production methods, which may be improved by a detailed understanding of taxol biosynthesis, especially the rate-limiting steps. The biosynthesis of taxol involves the cyclization of the common isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5), 11(2)-diene followed by extensive, largely oxidative, modification of this diterpene olefin. We set out to define the first oxygenation step in taxol biosynthesis. RESULTS: Microsomal enzymes from Taxus stem and cultured cells were used to define the first hydroxylation of taxadiene. We confirmed the structure of the reaction product (taxa-4(20), 11(12)-dien-5alpha-ol) by synthesizing this compound. The responsible biological catalyst was characterized as a cytochrome P450 (heme thiolate protein). In vivo studies confirmed that taxadienol is a biosynthetic intermediate and indicated that the hydroxylation step that produces this product is slow relative to subsequent metabolic transformations. CONCLUSIONS: The structure of the first oxygenated intermediate on the taxol pathway establishes that the hydroxylation reaction proceeds with an unusual double bond migration that limits the mechanistic possibilities for subsequent elaboration of the oxetane moiety of taxol. The reaction is catalyzed by a cytochrome P450, suggesting that the seven remaining oxygenation steps in taxol biosynthesis may involve similar catalysts. Because the first oxygenation step is slow relative to subsequent metabolic transformations, it may be possible to speed taxol biosynthesis by isolating and manipulating the gene for the taxadiene-5-hydroxylase that catalyzes this reaction.

Alkenes↗
PubMed1995

Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first committed step of taxol biosynthesis.

The first step in the biosynthesis of taxol in Pacific yew (Taxus brevifolia) is the cyclization of the universal diterpene precursor geranylgeranyl pyrophosphate to taxa-4(5),11(12)-diene. This parent olefin of the taxane diterpenoids is then elaborated to taxol and related compounds by a complex series of reactions involving oxidations and side-chain acylations. Cyclization activity is located principally in yew stem bark and adhering cambium. The operationally soluble cyclization enzyme was partially purified (approximately 600-fold) by combination of anion exchange, hydrophobic interaction, and dye-ligand chromatography. Nondenaturing, followed by denaturing, polyacrylamide gel electrophoresis, in combination with gel permeation chromatography, allowed the identification of taxadiene synthase as a monomeric protein of molecular weight 79,000. In general properties (divalent metal ion requirement, kinetic constants, molecular weight), the taxadiene synthase of Pacific yew is similar to the diterpene cyclase abietadiene synthase involved in resin acid biosynthesis in other gymnosperms. However, in pH optimum and response to inhibitors, these two diterpene cyclases are distinctly different. The activity (and enzyme protein) levels of Pacific yew taxadiene synthase are much lower than those for abietadiene synthase of lodgepole pine stem (constitutive) or of grand fir stem (wound-inducible) and the enzyme is not inducible to higher levels by stem wounding or elicitor treatment.

Alkenes↗
PubMed1995

Monoterpene synthases from gymnosperms and angiosperms: stereospecificity and inactivation by cysteinyl- and arginyl-directed modifying reagents.

To further define specific structural and mechanistic differences among monoterpene synthases from divergent plant sources, the stereospecificity of the enzyme-catalyzed isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which have been well established for monoterpene synthases from herbaceous angiosperms) were examined for monoterpene synthases from a conifer, lodgepole pine (Pinus contorta). The chiral monoterpenes isolated from lodgepole pine oleoresin and the major chiral products from cell-free assays of each of the four lodgepole pine monoterpene synthases belonged to the stereochemical family related by the biosynthetic intermediacy of 3S-linalyl pyrophosphate. Furthermore, both the putative intermediate, 3S-linalyl pyrophosphate, and the natural substrate, geranyl pyrophosphate, were enzymatically converted to the same monoterpene enantiomers. Thus, like monoterpene synthases from herbaceous angiosperms, monoterpene synthases from lodgepole pine appear to catalyze both the stereospecific isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound intermediate to multiple, stereochemically related monoterpene olefin isomers. The susceptibility of monoterpene synthases to inactivation by cysteinyl- and arginyl-directed chemical modification reagents was also examined to identify specific structural differences between enzymes from conifers and angiosperms. Like monoterpene synthases from peppermint (Mentha x piperita) and culinary sage (Salvia officinalis), monoterpene synthases from lodgepole pine were inactivated by thiol-directed reagents; however, unlike monoterpene synthases from these herbaceous angiosperms, monoterpene synthases from lodgepole pine were not protected against inactivation by coincubation with substrate and metal ion cofactor. Lodgepole pine monoterpene synthases were also inactivated by the arginyl-directed reagent phenylglyoxal, and coincubation with substrate and cofactor, to effect active-site protection, reduced the rate of inactivation 10-fold. (+)-Pinene synthase and (-)-pinene synthase from sage were also inactivated by phenylglyoxal, but no protection was afforded by coincubation with substrate and cofactor. Thus, monoterpene synthases of conifers appear to have catalytically important arginyl residues specifically located at or near the active site and have at least some catalytically important thiol residues at a non-substrate-protectable region of the enzyme, in contrast to monoterpene synthases from angiosperms which appear to have catalytically important cysteinyl residues at the active site and have catalytically important arginyl residues located at a non-substrate-protectable region of the enzyme.

Arginine↗
PubMed1995

Induced oleoresin biosynthesis in grand fir as a defense against bark beetles.

Grand fir (Abies grandis) saplings and derived cell cultures are useful systems for studying the regulation of defensive oleoresinosis in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced production of monoterpene olefins (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. The pathways and enzymes involved in monoterpene and diterpene resin acid biosynthesis are described, as are the coinduction kinetics following stem injury as determined by resin analysis, enzyme activity measurements, and immunoblotting. The effects of seasonal development, light deprivation, and water stress on constitutive and wound-induced oleoresinosis are reported. Future efforts, including a PCR-based cloning strategy, to define signal transduction in the wound response and the resulting gene activation processes are delineated.

Amino Acid Sequence↗
PubMed1995

Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene is the committed step of taxol biosynthesis in Pacific yew.

The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11(12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol.

Alkenes↗
PubMed1995

Isolation and characterization of an active-site peptide from a monoterpene cyclase labeled with a mechanism-based inhibitor.

(+)-Pinene synthase and (+)-bornyl pyrophosphate synthase from culinary sage (Salvia officinalis L.: Lamiaceae) catalyze the coupled isomerization and cyclization of geranyl pyrophosphate to the indicated bicyclic monoterpenes. The reaction parameters for these monoterpene cyclases have been well defined but the two enzymes have proved difficult to separate and purify in sufficient amounts for detailed structural characterization. A method was developed for the isolation of the two cyclases from highly enriched sage leaf oil gland extracts and for the efficient copurification of the two enzymes to about 95% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); the preparation yielded two overlapping species on two-dimensional polyacrylamide gel electrophoresis as expected. The cyclases were copurified and labeled with the highly selective mechanism-based irreversible inactivator 6-[1-3H]cyclopropylidene-3E-methyl-hex-2-en-1-yl pyrophosphate, subjected to cleavage with CNBr, and the resulting covalently modified peptides were isolated by SDS-PAGE for blotting to a polyvinylidene difluoride membrane and N-terminal amino acid sequence analysis. A radiochemically abundant 5-kDa peptide of the cleavage mixture was shown to be highly homologous, through 22 residues, to a segment (Leu197-Glu218) of (-)-4S-limonene synthase from spearmint (Mentha spicata L.: Lamiaceae), the only monoterpene cyclase for which the complete deduced amino acid sequence is known. These results illustrate the use of the mechanism-based inhibitor for purification and structural studies with the monoterpene cyclases, and they define a presumptive active site region that bears a highly conserved sequence among these enzymes from the mint (Lamiaceae) family.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri.

S-Linalool is one of the volatiles emitted by Clarkia breweri Grey [Green] flowers to attract its moth pollinator. S-Linalool synthase, the enzyme that stereoselectively converts the ubiquitous C10 intermediate GPP to S-linalool, is abundant in stigmata of freshly opened flowers, and it was purified to > 95% homogeneity by anion-exchange and hydroxyapatite chromatography. S-Linalool synthase is operationally soluble as are other monoterpene synthases, has a Km of 0.9 microM for geranyl pyrophosphate, exhibits a strict requirement for a divalent metal cofactor with a preference for Mn2+ (Km = 45 microM), and shows an optimal pH of 7.4. The enzyme is active as a monomer of 76 +/- 3 kDa as determined by gel permeation chromatography and polyacrylamide gel electrophoresis. Neither S- nor R-linalyl pyrophosphates are substrates for the C. breweri S-linalool synthase, although this tertiary allylic pyrophosphate ester is a bound intermediate in the biosynthesis of cyclic monoterpenes from geranyl pyrophosphate in many plant species, where it also serves as an alternate substrate.

Acyclic Monoterpenes↗
PubMed1995

Cytochrome P450 limonene hydroxylases of Mentha species.

The oxygenation pattern of the monoterpenoids of mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-limonene. In peppermint, C3-allylic hydroxylation leads to (-)-trans-isopiperitenol that ultimately is converted to (-)-menthol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol that is oxidized to (-)-carvone. The limonene-6-hydroxylase and the cytochrome P450 reductase were purified from the oil glands of spearmint, and the system was reconstituted. Amino acid sequences from the purified hydroxylase were utilized to design primers with which a large, non-degenerate PCR product was prepared. This probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated. This clone provides the tool for isolating the homologous cDNA species from peppermint and related Mentha species.

Cytochrome P-450 Enzyme System↗
PubMed1994

Diterpenoid resin acid biosynthesis in conifers: enzymatic cyclization of geranylgeranyl pyrophosphate to abietadiene, the precursor of abietic acid.

Abietic acid is a major component of the rosin fraction of oleoresin synthesized by conifer species, such as grand fir (Abies grandis) and lodgepole pine (Pinus contorta), as a defensive secretion against insect and pathogen attack. This diterpenoid resin acid is derived from geranylgeranyl pyrophosphate by conversion to abietadiene and sequential oxidation of the C18-methyl group of the precursor olefin to a carboxyl function. Resin acid biosynthesis is constitutively expressed at high levels in lodgepole pine stem and is induced to these levels by stem wounding in grand fir. Soluble enzyme extracts of lodgepole pine stem and of mechanically wounded grand fir stem catalyzed the divalent metal ion-dependent cyclization of [1-3H]geranylgeranyl pyrophosphate to (-)-abieta-7(8),13(14)-diene. The wound-inducible enzyme was partially purified and identified as an 80-kDa protein with general characteristics typical of those of other terpenoid cyclases. Although the enzymatic cyclization sequence almost certainly involves the formation of copalyl pyrophosphate and a pimaradiene as stable intermediates, no evidence for the separation of the corresponding partial activities was obtained. A pathway involving the production of various pimaradiene and abietadiene isomers is proposed to account for the origin of several common resin acids.

Abietanes↗
PubMed1994

Biosynthesis of monoterpenes: partial purification, characterization, and mechanism of action of 1,8-cineole synthase.

Geranyl pyrophosphate: 1,8-cineole cyclase (cineole synthase) catalyzes the conversion of geranyl pyrophosphate to the symmetrical monoterpene ether 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) by a process thought to involve the initial isomerization of the substrate to the tertiary allylic isomer, linalyl pyrophosphate, and cyclization of this bound intermediate to the alpha-terpinyl carbocation that is subsequently captured by water and undergoes heterocyclization to the remaining double bond. The enzyme was isolated from the secretory cells of the glandular trichomes of Salvia officinalis (garden sage) and partially purified, and the properties of this monoterpene cyclase, previously determined in crude cell-free extracts, were reexamined. These properties (pH optimum, divalent metal ion requirement, molecular weight, pI) were similar to those determined previously with the exception of substrate utilization; geranyl pyrophosphate was shown to be a more efficient substrate than the cis-isomer, neryl pyrophosphate, in the absence of competing phosphatase activity that contaminated earlier preparations of this enzyme. As with other monoterpene cyclases of herbaceous species, cineole synthase was inhibited by cysteine- and histidine-directed reagents, and protection against inactivation was provided by the substrate-metal ion complex. Studies with 18O-labeled acyclic precursors and H(2)18O, followed by mass spectrometric analysis of the product, confirmed that water was the sole source of the ether oxygen atom of 1,8-cineole. The electrophilic nature of the coupled isomerization-cyclization reaction was examined with a series of substrate and intermediate analogues. The overall stereochemistry of the cyclization of geranyl pyrophosphate to the symmetrical monoterpene was established by determining the enantioselectivity for (3R)- or (3S)-linalyl pyrophosphate as an alternative substrate and by oxidation of [3-3H]1,8-cineole, derived from [1-3H]geranyl pyrophosphate, to (+/-)-3-keto-1,8-cineole and radio-GLC separation of diastereomeric ketal derivatives to determine the labeled enantiomer.

Carbon-Carbon Lyases↗
PubMed1994

Monoterpene synthases of Pinus contorta and related conifers. A new class of terpenoid cyclase.

A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand fir (Abies grandis) and other higher plants in molecular mass (67 +/- 2 kDa as estimated by size-exclusion chromatography), Km for geranyl pyrophosphate (7.8 +/- 1.9 microM), and isoelectric point (4.75 +/- 0.2 as determined by isoelectric focusing), but the cyclases from both lodgepole pine and grand fir are unlike previously characterized terpenoid cyclases from angiosperms and fungi, in that they have an alkaline pH optimum (pH 7.8), are activated by K+, Rb+, Cs+, or NH+4 (Li+ and Na+ are not effective), require either Mn2+ or Fe2+ as divalent metal ion cofactors (Mg2+ is not effective), and are not protected by the substrate-metal ion complex against inhibition by the histidine-directed reagent diethyl pyrocarbonate. Chromatography of the pine xylem extracts on a quaternary amino anion-exchange resin results in the separation of four similar, but distinct, multiple product monoterpene synthases that produce sabinene, beta-phellandrene, 3-carene, and beta-pinene as the principal components, respectively. The major cyclase (phellandrene synthase) was subsequently purified by hydroxyapatite chromatography and electrophoresis. V8 proteolysis provided a peptide map significantly different from that obtained with limonene synthase from spearmint (Mentha spicata), and limited NH2-terminal sequencing of the phellandrene synthase fragments revealed no significant similarity to the deduced amino acid sequence of the angiosperm limonene synthase, the only monoterpene cyclase to be cloned and sequenced thus far. Furthermore, polyclonal antibodies raised against the angiosperm limonene synthase did not detectably cross-react with any proteins in extracts from either lodgepole pine or grand fir by immunoblotting analysis. In addition to these structural differences between cyclases from conifers and herbaceous angiosperms, the unusual pH optimum, mono- and divalent metal ion requirement, and reactivity toward histidine carbethoxylation indicate that monoterpene cyclases isolated from conifers may also have a different complement of active-site amino acid residues involved in substrate binding and catalysis than those of terpenoid cyclases previously isolated from angiosperms.

Cations, Divalent↗