Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase

Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).     

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.     

Isoprenyl diphosphate synthases

Isoprenyl diphosphate synthases catalyze consecutive condensations of isopentenyl diphosphates with allylic primer substrates to form linear backbones for all isoprenoid compounds including cholesterol. These synthases are classified according to the final chain length of their end products and the stereochemistry of the newly formed double bonds. Mutagenesis and X-ray crystallography data have uncovered the basic catalytic and chain length determination mechanisms of E-isoprenyl diphosphate synthases and shed light on their possible evolutionary course. Although much less is known about the Z-isoprenyl diphosphate synthase family, successful cloning and subsequent crystallizations in the near future will no doubt bring more insight as researchers begin to unravel the essential components and precise reaction mechanisms of this cellular machinery.     

Metabolism of deoxycytidine in isolated perfused organs

Summary The metabolism of deoxycytidine-U-¹⁴C was studied in isolated perfused normal and regenerating rat liver, in perfused rat intestine and in isolated perfused mouse liver. A technique to perfuse mouse liver is described. Catabolism of dCyd is very slow in organs of the rat whereas it is rapid in those of the mouse. Nevertheless, incorporation into liver DNA does not differ markedly between the species. The quantitative aspects of dCyd metabolism in the isolated organs are discussed.

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Zusammenfassung Der Stoffwechsel von Deoxycytidin-U-¹⁴C wurde in isoliert perfundierter normaler und regenerierter Rattenleber, in Rattendarm und in normaler Mäuseleber untersucht. Eine Methode zur Perfusion der Mäuseleber wird beschrieben. Der Abbau von Deoxycytidin ist langsam in Rattenorganen, jedoch rasch in Mäuseleber. Dabei sind zwischen den beiden Tierarten keine großen Unterschiede beim Einbau in Leber-DNA zu verzeichnen. Die quantitativen Aspekte des Deoxycytidinstoffwechsels in isolierten Organen werden diskutiert.

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Abbreviations used in text and figures dCyd deoxyoytidine - dThd deoxythymidine - dUrd deoxyuridine - dCMP deoxyoytidine monophosphate - BAIBA ß-aminoisobutyrio acid This is publication No. 754 of the Euratom Biology Division, Contract 078-69 BIAC.     

Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase

Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

5-Fluorocytosine derivatives as inhibitors of deoxycytidine kinase

A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.     

Analysis of deoxycytidine accumulation in gemcitabine treated patients

Deoxycytidine (CdR) analogs are increasingly popular as chemotherapeutic agents and their effectiveness can be linked to the direct competition with active forms of endogenous CdR. A tandem mass spectrometric assay was developed to determine the plasma concentrations of CdR. Plasma extracts were prepared by protein precipitation and an ethyl acetate/water back extraction, and then separated chromatographically. Detection parameters were optimized for multi-reaction monitoring (MRM) tandem mass spectrometry and assay efficiency was improved using 15N3 CdR as an isotopic internal standard. Preliminary results from a gemcitabine trial are shown which indicate that CdR concentrations increase systemically during infusion, from about 5 nM to 78 nM after hepatic artery infusion and to 102 nM after systemic infusion for 24 hours. The developed assay demonstrated good sensitivity and selectivity for CdR.