Preparation Of Synthase Research Articles

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Methods for the preparation and analysis of a bifunctional class II diterpene synthase, copalyl diphosphate synthase from Penicillium fellutanum.

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.

Preparation and self-cleavage of fusion soluble farnesyl diphosphate synthase in E. coli

Farnesyl diphosphate synthase (FPPS) is a crucial protein in terpenoid production. However, its industrial application is limited owing to its low solubility in Escherichia coli. In this study, we focused on ispA encoding FPPS and designed a fusion expression system to reduce inclusion body (IB) formation. Among the chosen fusion tags, the GB1-domain (GB1) exhibited the highest ability to solubilize the recombinant protein. Increased rare tRNA abundance not only improved the GB1-FPPS yield but also increased its soluble level. A “one-step” method for the acquisition of soluble FPPS was also considered. By combining GB1-FPPS expression and Tobacco Etch Virus protease (TEVp) cleavage in vivo, a controllable GB1-FPPS “self-cleavage” system was constructed. Overall, this study provides an efficient approach for obtaining soluble forms of FPPS, which show great potential for use in the soluble expression of other homologous diphosphate synthase.

Preparation of a whole cell catalyst overexpressing acetohydroxyacid synthase of Thermotoga maritima and its application in the syntheses of \u03b1-hydroxyketones

The large catalytic subunit of acetohydroxyacid synthase (AHAS, EC 2.2.1.6) of Thermotoga maritima (TmcAHAS) was prepared in this study. It possesses high specific activity and excellent stability. The protein and a whole cell catalyst overexpressing the protein were applied to the preparation of α-hydroxyketones including acetoin (AC), 3-hydroxy-2-pentanone (HP), and (R)-phenylacetylcarbinol (R-PAC). The results show that AC and HP could be produced in high yields (84% and 62%, respectively), while R-PAC could be synthesized in a high yield (about 78%) with an R/S ratio of 9:1. Therefore, TmcAHAS and the whole cell catalyst overexpressing the protein could be practically useful bio-catalysts in the preparation of α-hydroxyketones including AC, HP, and R-PAC. To the best of our knowledge, this is the first time that bacterial AHAS was used as a catalyst to prepare HP with a good yield, and also the first time that TmcAHAS was employed to synthesize AC and R-PAC.

Concise route to the chiral pyrrolidine core of selective inhibitors of neuronal nitric oxide.

2-(((3R,4R)-4-(Allyloxy)-1-benzylpyrrolidin-3-yl)methyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (2), a key intermediate for the preparation of novel neuronal nitric oxide synthase (nNOS) inhibitors, is synthesized using diisopropyl (R)-(+)-malate as the starting material. The key steps involve a Frater-Seebach diastereoselective alkylation and a fast intramolecular cyclization.

Preparation, Crystallization and Preliminary X-Ray Analysis of Threonine Synthase from Streptococcus mutans

The thrC gene of Streptococcus mutans encodes threonine synthase, which is a potential target for drug design. To study the structure and function of the enzyme, the thrC gene was amplified from Streptococcus mutans genomic DNA and cloned into the expression vector pET28alpha. The protein was expressed in Escherichia coli in soluble form and purified to homogeneity. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method. The crystal diffracted to 2.5 A and belonged to space group P3(1) or P3(2), with unit cell parameters a=b=60.39 A, c=118.62 A.

Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) fromStreptococcus mutans

The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni(2+)-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 A resolution. The crystal belongs to space group P2(1), with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 A, beta = 94.55 degrees .

Production of Nitric Oxide by Mitochondria

The production of NO. by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio = 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-FeII produced a signal ascribed to the NO. spin adduct (g = 2.04; aN = 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor NG-monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO. (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO. x (min.mg protein)-1, respectively, with a Km for L-Arg of 5-7 microM. Comparison of the rates of NO. production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO., the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation.

Bromoacetamido analogs of indomethacin and mefenamic acid as affinity-labeling agents and mechanistic probes for prostaglandin H2 synthase.

Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene.

A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury.

Preparation and assay of chalcone synthase from walnut tree tissue

Chalcone synthase activity was obtained from bark tissue of one-year-old shoots taken from a 30-year-old hybrid walnut tree Juglans nigra §x J. regia. High tissue concentrations of phenolics, which are a major problem in woody plant enzyme extraction, were essentially overcome by the use of 200 mM ascorbic acid, 1.5% (w/v) polyethyleneglycol and 50 mM cysteine in the buffers, and a constant flow of nitrogen. Some properties of walnut tree chalcone synthase are discussed in comparison to those of the same enzyme from other plants.

Purification and Characterization of Dihydrodipicolinate Synthase from Pea

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-beta-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-beta-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-alpha-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-alpha-(2-aminoethoxyvinyl)glycine is noncompetitive towards l-aspartate-beta-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-beta-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.

Concerted loss of cyclooxygenase and peroxidase activities from prostaglandin H synthase upon proteolytic attack

Prostaglandin H synthase has two distinct enzymatic activities: a cyclooxygenase that forms PGG2 from arachidonate and a peroxidase that can reduce hydroperoxidases, such as PGG2, to the corresponding alcohols. The relative sensitivities of the two synthase activities to proteolytic attack have been examined, using trypsin, chymotrypsin, and proteinase K, all known to attack the native apoprotein in the arg 253 region. The relation between the specific activity of the synthase and the loss of the two activities and the cleavage of the synthase subunit during trypsin digestion was also examined. The cyclooxygenase and peroxidase activities declined in concert throughout room temperature digestions with each of the three proteases. There was no indication of a selective loss of either activity in any of the digestions. In separate digestions with the same preparation of synthase, 3.3% (w/w) proteinase K resulted in more extensive loss of activity (90% decrease after 90 min) than did 3% (w/w) trypsin (70% decrease after 120 min) or 5% (w/w) chymotrypsin (60% decrease after 135 min). In tryptic digestions of synthase preparations with cyclooxygenase specific activity between 16 and 125 k units/mg protein, the fractional loss of cyclooxygenase activity was, within experimental error, the same as that of peroxidase activity. The extent of cleavage of the 70 kDa synthase subunit was greater than the loss of enzymatic activity, with the discrepancy being larger for synthase preparations with lower specific activity. The presence of a variable amount of catalytically-inactive, protease-sensitive, synthase protein could account for the difference between surviving activity and intact subunit in six out of the seven synthase preparations examined. Thus, it is likely that the cyclooxygenase and peroxidase activities are destroyed together during proteolytic attack on the arg 253 region of the native synthase apoprotein.

A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin

Rabbit skeletal muscle glycogen previously has been shown to be covalently bound to a 40,000-Da protein (“glycogenin”) via a novel glucosyl-tyrosine linkage [ I. R. Rodriguez and W. J. Whelan (1985) Biochem. Biophys. Res. Commun. 132, 829–836 ]. Antibodies raised against rabbit skeletal muscle glycogenin cross-react with a similar protein present in rabbit heart and liver glycogens, as well as with a 42,000-Da “acceptor protein” present in high-speed supernatants of rabbit muscle, heart, retina, and liver. This 42,000-Da protein incorporates [U- 14C]Glc when an ammonium sulfate fraction prepared from the tissue supernatants is incubated with UDP-[U- 14C]Glc. The [U- 14C]-Glc incorporated can be removed quantitatively by treatment with amylolytic enzymes, indicating that the [U- 14C]Glc incorporation represents elongation of a preexisting glucan attached to the acceptor protein. Furthermore, a commercial preparation of rabbit skeletal muscle glycogen synthase contains this 42,000-Da protein. We propose that the 42,000-Da protein represents the free form of glycogenin in tissues, with its covalently attached glucan chain(s) providing a “primed” elongation site for glycogen synthesis.

Arginine decarboxylase is a component activity of the multifunctional enzyme putrescine synthase in cucumber seedlings

A homogenous PreParation of Putrescine synthase, the versatile multifunctional enzyme involved in agmatine →Putrescine conversion inCucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the Purified arginine decarboxylase mediated the comPonent as well as the comPlete set of couPled reactions harboured by Putrescine synthase. Both the enzyme PreParations exhibited identical electroPhoretic and chromatograPhic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional Putrescine synthase inCucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterPart inLathyrus sativus.

Improved preparation and assay of chalcone synthase

Preparation and assay of chalcone synthase in presence of sodium ascorbate and exclusion of oxygen during some steps gives improved yield and purity of 2 S-naringenin.

The mechanism of alkyldihydroxyacetone-P synthase. Formation of [3H]H2O from acyl[1-R-3H]dihydroxyacetone-P by purified alkyldihydroxyacetone-P synthase in the absence of acylhydrolase activity.

Alkyldihydroxyacetone-P (alkyl-DHAP) synthase catalyzes the exchange of the fatty acid esterified to C-1 of the DHAP portion of acyl-DHAP for a fatty alcohol to form 1-O-alkyl-DHAP, the first ether-linked intermediate in ether lipid biosynthesis. Another characteristic of the reaction is the exchange of the pro-R hydrogen at C-1. We have investigated this hydrogen exchange using palmitoyl-[1-R-3H]DHAP and a 1000-fold purified preparation of alkyl-DHAP synthase. We found a small but significant pro-R hydrogen exchange in the absence of the co-substrate, fatty alcohol. When [14C]hexadecanol was added, the increase in pro-R 3H exchange was equal to the [14C]hexadecyl-DHAP formed. Addition of [14C]palmitic acid resulted in an increase in pro-R 3H exchange that matched the formation of [14C]palmitoyl-DHAP by the acyl exchange activity of alkyl-DHAP synthase. Furthermore, although whole microsomes contain at least two acyl hydrolases for acyl-DHAP, purified preparations of alkyl-DHAP synthase do not form DHAP from acyl-DHAP. These results are discussed with respect to data obtained from other laboratories using whole microsomes and in support of our proposed ping-pong mechanism for alkyl-DHAP synthase.

Metabolism and activation of 7,8-dihydrobenzo[a]pyrene during prostaglandin biosynthesis. Intermediacy of a bay-region epoxide.

A Tween 20-solubilized preparation of prostaglandin endoperoxide synthase has been shown to metabolize 7,8-dihydrobenzo[a]pyrene (H2BP) to a form highly mutagenic to Salmonella typhimurium strain TA98. The arachidonic acid-dependent metabolism of H2BP by microsomal and purified prostaglandin endoperoxide synthase has been studied and the products identified. A spectral investigation of the metabolism indicated the bay-region double bond as the primary site of metabolism. Radiolabeled H2BP was synthesized and incubated with the enzyme preparations and the metabolites were separated by reverse phase high performance liquid chromatography and quantitated by liquid scintillation counting. Radioactive products were characterized by co-chromatography with chemically synthesized standards, UV-visible spectra, and mass spectrometry of acetate derivatives. The major polar products were determined to be trans- and cis-9,10-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8,9,10-tetrahydrobenzo[a]pyrene-9-one in a ratio of 1:1.2:0.4. The inclusion of 5 mM 3,3,3-trichloropropene-1,2-oxide, an epoxide hydrolase inhibitor, produced the same products but in a ratio of 1:2.3:1.2. Incubations with purified prostaglandin endoperoxide synthase yielded the three products in a ratio of 1:2.8:0.7. The major nonpolar product was identified as benzo[a]pyrene. The polar products of metabolism, the effects of 3,3,3-trichloropropene-1,2-oxide on their distribution, and the detection of a mutagenic intermediate support the conclusion that H2BP is co-oxygenated during prostaglandin biosynthesis to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

Isolation of the mature subunit of delta-aminolaevulinate synthase from embryonic chick liver.

We presented evidence indicating that the established procedure for purifying delta-aminolaevulinate (ALA) synthase from embryonic-chick liver yielded an enzyme with a partially degraded subunit of molecular weight 51000 [Ades & Harpe (1981) J. Biol. Chem. 256, 9329-9333]. We now report the purification from livers of porphyric embryos of a preparation of ALA synthase which consisted primarily of a 63000-Da polypeptide and a component migrating as a smear of polypeptides with a minimum molecular weight of 52 000. Neither component could be recovered from liver mitochondria of normal embryos, where the amounts of ALA synthase were relatively low. The 52 000-Da component had been established to be the partially degraded subunit of the enzyme. Peptide-mapping analyses indicated that the 63 000- and the 52 000-Da components possessed significant structural homologies, and it was concluded that the 63 000-Da polypeptide represented the mature subunit of ALA synthase.

14] Preparation and assay of prostacyclin synthase

This chapter presents procedure for preparation and assay of prostacyclin synthase. In purification procedure described in the chapter batches of about 500 g of frozen tissue were thawed, and the vascular adventitia was removed. The unseparated aortic walls were cut into pieces, then rapidly frozen in liquid nitrogen and immediately pulverized into a fine powder. The powder was suspended in 0.05 M Tris buffer at pH 7.5 and homogenized at high speed using a Polytron homogenizer. Finally, 10,000 g supernatant was centrifuged in a refrigerated ultracentrifuge to obtain a pellet termed the microsomal fraction. The prostacyclin synthase activity resides primarily in this fraction, and usually resuspends in deionized water and then lyophilize. Prostacyclin is unstable, with a half-life of approximately 10 min at physiological temperature and pH; it hydrolyzes quantitatively and nonenzymically to 6-keto-PGF 1α . Only bioassays permit the assay of prostacyclin itself in biological samples, and therefore the majority of investigators have sought to assess prostacyclin synthase by measuring the formation of 6-keto-PGF lα by radiochemical, radioimmunoassay, or physicochemical procedures.