Purification and characterization of two forms of geranyl transferase from Ricinus communis

Green, Terrence R.; West, Charles A.

doi:10.1021/bi00720a007

Cited by 34 publications

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“…Separation ofGeranyl and Farnesyl Transferase by Sievorptive Chromatography. Previous work demonstrated that FPP is synthesized in castor bean seedling extracts from IPP and DMAPP or GPP by geranyl transferases (8). The evidence-cited above in this paper suggests that a separate farnesyl transferase is responsible for the elongation of FPP to GGPP in castor bean seedling extracts.…”

supporting

confidence: 52%

“…As Figure 5 shows, geranyl transferase activity is separated from the farnesyl transferase activity by this procedure. ['4C]Farnesol and [14C]geranylgeraniol produced enzymically from the products of the geranyl transferase and famesyl transferase reactions, respectively, were identified by co-chromatography with authentic samples by reverse phase chromatography (8). These results indicate that the purification step is sufficient to separate geranyl transferases from farnesyl transferase.…”

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confidence: 93%

“…The enzyme farnesyl pyrophosphate synthetase, which catalyzes two successive prenyl transferase reactions with DMAPP and GPP as the allylic substrates, has been intensively investigated from fungal and animal sources (16). Geranyl transferases have been partially purified and characterized from plant sources (1,14,24), including castor bean seedlings (8). Geranylgeranyl pyrophosphate synthetase, which catalyzes a famesyl transferase reaction with FPP as the allylic substrate, and may also catalyze transferase reactions with DMAPP and GPP as the allylic substrates in some cases, has been examined from only a few higher plant sources including pumpkin seedlings (15,19), tomato fruit plastids (21), and carrot cells (13 butanediol succinate on Anakrom SD 80/90 mesh in a Varian 90-P gas chromatograph.…”

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Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Dudley¹,

Green²,

West³

1986

Plant Physiol.

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ABSTRACIFarnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg2" ion at 4 millimolar gave the greatest stimulation of activity; Mn2 ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (Km = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K,,, = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K. = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes-a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.Casbene, a macrocyclic diterpene hydrocarbon with antifungal antibiotic properties, is produced in cell-free extracts of castor bean seedlings which have been exposed to spores of Rhizopus stolonifer or other fungi (20), to the extracellular endopolygalacturonase of R. stolonifer culture filtrates (12), or to pectic fragments released from pectic substances of the plant cell wall

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confidence: 93%

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Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Dudley¹,

Green²,

West³

1986

Plant Physiol.

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“…Other studies have shown that many ,E,E-FPP synthases accept both DMAPP and GPP as allylic substrates (25)(26)(27)(28)(29)(30). In cases in which ,E,E-FPP is synthesized from IPP and DMAPP by a single enzyme, two condensations of IPP with the allylic substrate are catalyzed, releasing only small amounts of the GPP intermediate (31).…”

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confidence: 99%

Identification of a novel class of ω,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis

Dhiman

Schulbach

Mahapatra

et al. 2004

Journal of Lipid Research

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We have identified an ,E,E -farnesyl diphosphate ( ,E,E -FPP) synthase, encoded by the open reading frameRv3398c , from Mycobacterium tuberculosis that is unique among reported FPP synthases in that it does not contain the type I (eukaryotic) or the type II (eubacterial) ,E,E -FPP synthase signature motif. Instead, it has a structural motif similar to that of the type I geranylgeranyl diphosphate synthase found in Archaea . Thus, the enzyme represents a novel class of ,E,E -FPP synthase. Rv3398c was cloned from the M. tuberculosis H37Rv genome and expressed in Mycobacterium smegmatis using a new mycobacterial expression vector (pVV2) that encodes an in-frame N-terminal affinity tag fusion with the protein of interest. The fusion protein was well expressed and could be purified to near homogeneity, allowing facile kinetic analysis of recombinant Rv3398c. Of the potential allylic substrates tested, including dimethylallyl diphosphate, only geranyl diphosphate served as an acceptor for isopentenyl diphosphate. The enzyme has an absolute requirement for divalent cation and has a K m of 43 M for isopentenyl diphosphate and 9.8 M for geranyl diphosphate and is reported to be essential for the viability of M. tuberculosis. ,E,E -farnesyl diphosphate ( ,E,E -FPP) is typically synthesized by short-chain isoprenyl diphosphate synthases that catalyze an electrophilic alkylation reaction of isopentenyl diphosphate (IPP) to an allylic diphosphate such as dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) ( Fig. 1 ). To date, amino acid sequence alignments have been generated for more than 50 E -isoprenyl diphosphate synthases from diverse sources (1-3). These studies have identified five regions of amino acid conservation, including two aspartate-rich motifs that are referred to as the first and second aspartate-rich motifs. The motifs are necessary for binding the diphosphate moieties of the substrates (4), and site-directed mutagenesis experiments have defined key amino acids around the first aspartate-rich motif (FARM) that determine the chain length of the product (5-8). These amino acids constitute the chain length-determining (CLD) region.In a recent review, the CLD region and the FARM signatures were used to segregate the ,E,E -FPP synthases into two classes designated type I (eukaryotic) and type II (eubacterial) (1). Both of these classes of enzyme presumably evolved from a common precursor similar to the archaeal type I geranylgeranyl diphosphate (GGPP) synthase, which contains the FARM signature DDXXD and a single aromatic amino acid in the fifth position N-terminal to the FARM. Type I ,E,E -FPP synthases (eukaryotic) are similar to the type I GGPP synthases in that they also contain the DDXXD motif. However, they have two aromatic amino acids at the fourth and fifth positions N-terminal to the FARM. Type II ,E,E -FPP synthases (eubacterial) contain the FARM signature DDXXXXD and a single aromatic amino acid in the fifth position N-terminal to the FARM. This classification system is now well e...

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“…The postulated intermediates and enzymes involved in the biosynthesis of casbene from MVA are illustrated in Figure 1. Two geranyl transferase isoenzymes (11), farnesyl transferase (6), and casbene synthetase (7) Mg protein/ml). Preliminary studies indicated that the reaction rate was linear with this range of enzyme concentrations for 5 min at 30°C.…”

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Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Dudley¹,

Dueber²,

West³

1986

Plant Physiol.

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ABSTRACICasbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.

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Purification and characterization of two forms of geranyl transferase from Ricinus communis

Cited by 34 publications

References 23 publications

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Identification of a novel class of ω,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

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