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PubMed1998

Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production.

Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction. In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T. canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.

Acetates↗
PubMed1992

Biosynthesis of trans,trans,trans-geranylgeranyl diphosphate by the cytosolic fraction from rat tissues.

The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.

Animals↗
PubMed1998

Administration of fructose 1,6-diphosphate during early reperfusion significantly improves recovery of contractile function in the postischemic heart.

OBJECTIVES: Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. METHODS: Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37 degrees C followed by reperfusion (n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31-nuclear magnetic resonance spectroscopy. RESULTS: In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 +/- 2 vs 70 +/- 4 mm Hg, p < 0.05), lower diastolic pressures (7 +/- 1 vs 12 +/- 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. CONCLUSIONS: We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion.

Adenosine Triphosphate↗
PubMed2003

An alternative mechanism of product chain-length determination in type III geranylgeranyl diphosphate synthase.

(All-E) prenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphates with allylic prenyl diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl diphosphate synthases, i.e. farnesyl diphosphate synthases and geranylgeranyl diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III geranylgeranyl diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl diphosphate synthases.

Alkyl and Aryl Transferases↗
PubMed1995

Photoreactive analogues of prenyl diphosphates as inhibitors and probes of human protein farnesyltransferase and geranylgeranyltransferase type I.

Photoreactive analogues of prenyl diphosphates have been useful in studying prenyltransferases. The effectiveness of analogues with different chain lengths as probes of recombinant human protein prenyltransferases is established here. A putative geranylgeranyl diphosphate analogue, 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate (DATFP-FPP), was the best inhibitor of both protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PFFT-I). Shorter photoreactive isprenyl diphosphate analogues with geranyl and dimethylallyl moieties and the DATFP-derivative of farnesyl monophosphate were much poorer inhibitors. DATFP-FPP was a competitive inhibitor of both PFT and PGGT-I with Ki values of 100 and 18 nM, respectively. [32P]DATFP-FPP specifically photoradiolabelled the beta-subunits of both PFT and PGGT-I. Photoradiolabelling of PGGT-I was inhibited more effectively by geranylgeranyl diphosphate than farnesyl diphosphate, whereas photoradiolabelling of PFT was inhibited better by farnesyl diphosphate than geranylgeranyl diphosphate. These results lead to the conclusions that DATFP-FPP is an effective probe of the prenyl diphosphate binding domains of PFT and PGGT-I. Furthermore, the beta-subunits of protein prenyltransferases must contribute significantly to the recognition and binding of the isoprenoid substrate.

Affinity Labels↗
PubMed1996

A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product.

Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.

Alkyl and Aryl Transferases↗
PubMed2005

Disruption of the structural gene for farnesyl diphosphate synthase in Escherichia coli.

The chromosomal ispA gene encoding farnesyl diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of farnesyl diphosphate was decreased. The condensation of isopentenyl diphosphate with dimethylallyl diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of farnesyl diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl diphosphate synthase or undecaprenyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed1997

Evaluation of a novel lipid prodrug for intraocular drug delivery: effect of acyclovir diphosphate dimyristoylglycerol in a rabbit model with herpes simplex virus-1 retinitis.

BACKGROUND: Acyclovir diphosphate dimyristoylglycerol is a lipid prodrug of acyclovir that forms liposomes and provides substantial activity against herpes simplex virus, acyclovir-resistant strains of herpes simplex virus, and human cytomegalovirus. We therefore tested this promising new drug in a rabbit model of herpes simplex retinitis. METHODS: A total of 22 pigmented rabbits were pretreated with either acyclovir diphosphate dimyristoylglycerol, ganciclovir, acyclovir, or buffer. Retinae then were inoculated with herpes simplex virus-1 or buffer 1 week after the injection of drug. In another experiment we compared the effects of acyclovir diphosphate dimyristoylglycerol and acyclovir diphosphate dioleoylglycerol on the optical clarity of vitreous. RESULTS: Animals injected intravitreally with acyclovir diphosphate dimyristoylglycerol showed retinitis that was less severe than that in animals injected with ganciclovir, acyclovir, and buffer; differences in grading scores of the retinitis between animals injected with acyclovir diphosphate dimyristoylglycerol and those injected with buffer were statistically significant (P = 0.0015). Vitreous and optical media became clear 4 days after acyclovir diphosphate dioleoylglycerol injection compared with 10 days after with acyclovir diphosphate dimyristoylglycerol injections. CONCLUSION: Acyclovir diphosphate dimyristoylglycerol had prolonged antiviral activity against herpes simplex virus-1 retinitis in a rabbit model. This drug delivery system, modified to improve optical clarity, may allow long-acting intravitreal treatment of cytomegalovirus retinitis and other retinal diseases.

Acyclovir↗
PubMed1975

Loss of Ribulose 1,5-Diphosphate Carboxylase and Increase in Proteolytic Activity during Senescence of Detached Primary Barley Leaves.

Symptoms typical of senescence occurred in green detached primary barley (Hordeum vulgare L.) leaves placed in darkness and in light. Chlorophyll, total soluble protein, ribulose 1,5-diphosphate carboxylase protein and activity each progressively decreased in darkness and to a lesser extent in light. In all treatments most of the total soluble protein lost was accounted for by a decrease in ribulose 1,5-diphosphate carboxylase protein, suggesting that the chloroplast was a major site of degradation early in senescence.Loss of ribulose 1,5-diphosphate carboxylase protein was negatively correlated with an increase in proteolytic activity measured against azocasein. Both rates were exponential, with about a 30% difference in apparent rate constants. Cycloheximide essentially prevented the loss of chlorophyll, ribulose 1,5-diphosphate carboxylase protein, and activity and completely inhibited the increase in proteolytic activity against azocasein. Since chloramphenicol had little effect on the loss of ribulose 1,5-diphosphate carboxylase protein or chlorophyll, or on proteolytic activity against azocasein, it is suggested that the proteolytic activity was developed on cytoplasmic 80 S ribosomes.Kinetin greatly retarded the onset of such symptoms of senescence by inhibiting the losses of chlorophyll and ribulose 1,5-diphosphate carboxylase protein and protected against inactivation of enzymic activity. It also prevented the increase in proteolytic activity measured against azocasein. Incorporation of labeled amino acids into ribulose 1,5-diphosphate carboxylase during its rapid degradation showed that the enzyme was under turnover. The changes in ribulose 1,5-diphosphate carboxylase protein and activity, chlorophyll, soluble protein other than ribulose 1,5-diphosphate carboxylase, proteolytic and esterolytic activity during senescence indicate that senescence is a selective, sequential process.

Journal Article↗
PubMed1983

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

Animals↗
PubMed2001

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as a secondary metabolite from D-fructose-1,6-diphosphate metabolism by Zygosaccharomyces rouxii.

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is an important aroma compound found in many fruits such as strawberries and pineapples and it is also produced by the soy-sauce-fermenting yeast Zygosaccharomyces rouxii after the addition of d-fructose-1,6-diphosphate to yeast-peptone-dextrose nutrient media. Dilute DMHF solutions exhibit a strawberry-like flavor while DMHF concentrates have a caramel-like aroma. In media containing D-fructose-1,6-diphosphate as the sole carbon source, growth of Z. rouxii and formation of DMHF were not observed. Although Z. rouxii cells grew in media with D-glucose as the sole carbon source, DMHF was only produced when media were supplemented with D-fructose-1,6-diphosphate. The DMHF concentration always correlated with the yeast cell count and D-fructose-1,6-diphosphate concentration. Addition of CaCl2 (up to 50 g.l(-1)) led to a higher DMHF concentration. Addition of Na2SO3 reduced the growth of Z. rouxii and inhibited DMHF formation. The amount of DMHF formed by Z. rouxii was not significantly affected by the addition of KH2PO4. DMHF concentrations of 5 and 10 g.l(-1) partially and completely inhibited the growth of Z. rouxii cells, respectively. Only the singly labeled furanone was formed after the addition of 1-13C-D-fructose-1,6-diphosphate to the medium. However, unlabeled DMHF was formed in the presence of (13)C(6)-D-glucose. Therefore, the carbons of the furanone originate exclusively from exogenously supplied D-fructose-1,6-diphosphate as no exchange with the internal pool of D-fructose-1,6-diphosphate occurs. This implies that DMHF is a secondary metabolite of Z. rouxii formed from D-fructose-1,6-diphosphate. We assume that at least the first step of the metabolism of D-fructose-1,6-diphosphate takes place in the cell wall or membrane of the yeast.

Journal Article↗
PubMed1994

Archaebacterial ether-linked lipid biosynthetic gene. Expression cloning, sequencing, and characterization of geranylgeranyl-diphosphate synthase.

Archaebacterial Sulfolobus acidocaldarius geranylgeranyl-diphosphate (GGPP) synthase (EC 2.5.1.29) catalyzes consecutive condensations of isopentenyl diphosphate with allylic diphosphates to produce GGPP which is the important precursor of archaebacterial ether-linked lipids. We developed an expression screening method for cloning the GGPP synthase gene, which utilizes the carotenoid biosynthesis genes of Erwinia uredovora to visualize a clone expressing GGPP synthase, and then screened a genomic DNA library from S. acidocaldarius for the GGPP synthase gene by using this method. Positive clones were shown to contain GGPP synthase gene by the use of an in vitro assay. Extracts from Escherichia coli transformants catalyzed the condensation of isopentenyl diphosphate with farnesyl diphosphate (FPP) to produce (all-E)-GGPP. The nucleotide sequence of the 2.3-kilobase HindIII fragment of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2. ORF-1 encodes GGPP synthase with the expected molecular weight of 36,873, and ORF-2 encodes a protein with homology for UDP-N-acetylglucosaminedolichyl phosphate N-acetylglucosaminephosphotransferase. The cloned GGPP synthase was partially purified with several chromatographies after heat treatment of cell free extract. This enzyme is extremely thermostable and has an optimal pH at 5.8. Dimethylallyl diphosphate, geranyl diphosphate, and (all-E)-FPP are, in decreasing order of activity, acceptable as allylic substrates to produce (all-E)-GGPP. When dimethylallyl diphosphate or geranyl diphosphate are the allylic substrates, a significant amount of mixture of the products is shorter than GGPP. (2Z,6E)-FPP is not a substrate. This enzyme recognizes the E-configuration of allylic substrate.

Alkyl and Aryl Transferases↗
PubMed2005

Functional coupling between nucleoside diphosphate kinase of the outer mitochondrial compartment and oxidative phosphorylation.

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.

Adenosine Diphosphate↗
PubMed2001

Increased activity of membrane-associated nucleoside diphosphate kinase and inhibition of cAMP synthesis in failing human myocardium.

OBJECTIVE: Chronic heart failure is associated with a decreased responsiveness of the heart to beta-adrenergic receptor agonists. We recently demonstrated a receptor-independent activation of G proteins and modulation of cardiac adenylyl cyclase activity by sarcolemmal membrane-associated nucleoside diphosphate kinase. We wondered whether changes in the activity of nucleoside diphosphate kinase occur in heart failure and contribute to or compensate for the impairment in myocardial receptor-mediated cAMP generation. METHODS: Sarcolemmal membranes were purified from non-failing and failing human left ventricular myocardium. The protein level and activity of nucleoside diphosphate kinase were quantified. The influence of nucleoside diphosphate kinase on adenylyl cyclase activity was determined by measuring the effect of GDP on adenylyl cyclase activity in the absence and presence of nucleoside diphosphate kinase inhibitors. RESULTS: The amount and activity of nucleoside diphosphate kinase in sarcolemmal membranes from failing hearts (n=13) were increased 3- to 4-fold compared to levels in membranes from non-failing myocardium (n=5). This increase in sarcolemmal nucleoside diphosphate kinase activity resulted in a 50% inhibition of adenylyl cyclase activity over a range of GDP and ATP concentrations. CONCLUSION: The amount and activity of nucleoside diphosphate kinase are increased in sarcolemmal membranes of failing human myocardium, resulting in a substantial receptor-independent inhibition of adenylyl cyclase activity.

5'-Nucleotidase↗
PubMed2006

Biochemical characterization of the decaprenyl diphosphate synthase of Rhodobacter sphaeroides for coenzyme Q10 production.

Coenzyme Q(10) (CoQ(10)), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells' survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ(10) production, as indicated by the amount of CoQ(9) formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, omega,E,E-farnesyl diphosphate (FPP), and omega,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of V (max) (pmol/min), K (M) (microM), k (cat) (1/min), and k (cat) /K (M) of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.

Agrobacterium tumefaciens↗
PubMed1983

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase.

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.

Diphosphates↗
PubMed1994

Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure.

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.

Alkyl and Aryl Transferases↗
PubMed1997

Polyprenyl diphosphate synthases.

It is noteworthy that in spite of the similarity of the reactions catalyzed by these prenyltransferases, the modes of expression of catalytic function are surprisingly different, varying according to the chain length and stereochemistry of reaction products. These enzymes are summarized and classified into four groups, as shown in Figure 13. Short-chain prenyl diphosphates synthases such as FPP and GGPP synthases require no cofactor except divalent metal ions, Mg2+ or Mn2+, which are commonly required by all prenyl diphosphate synthases. Medium-chain prenyl diphosphate synthases, including the enzymes for the synthesis of all-E-HexPP and all-E-HepPP, are unusual because they each consist of two dissociable dissimilar protein components, neither of which has catalytic activity. The enzymes for the synthesis of long-chain all-E-prenyl diphosphates, including octaprenyl (C40), nonaprenyl-(C45), and decaprenyl (C50) diphosphates, require polyprenyl carrier proteins that remove polyprenyl products from the active sites of the enzymes to maintain efficient turnovers of catalysis. The enzymes responsible for Z-chain elongation include Z,E-nonaprenyl-(C45) and Z,E-undecaprenyl (C55) diphosphate synthases, which require a phospholipid. The classification of mammalian synthases seems to be fundamentally similar to that of bacterial synthases except that no medium-chain prenyl diphosphate synthases are included. The Z-prenyl diphosphate synthase in mammalian cells is dehydrodolichyl PP synthase, which catalyzes much longer chain elongations than do bacterial enzymes. Dehydrodolichyl PP synthase will be a major target of future studies in this field in view of its involvement in glycoprotein biosynthesis.

Alkyl and Aryl Transferases↗