In Vivo Characterization of Phosphotransferase-Encoding Genes istP and forP as Interchangeable Launchers of the C3',4'-Dideoxygenation Biosynthetic Pathway of 1,4-Diaminocyclitol Antibiotics.

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

Biosynthesis of (deoxy)guanosine-5'-triphosphate by GMP kinase and acetate kinase fixed on the surface of E. coli.

(Deoxy)guanosine-5'-triphosphate (5'-(d)GTP), the precursor for synthesizing DNA or RNA in vivo, is an important raw material for various modern biotechnologies based on PCR. In this study, we investigated the application of whole-cell catalysts constructed by bacterial cell surface display in biosynthetic reactions of 5'-(d)GTP from (deoxy)guanosine-5'-monophosphate (5'-(d)GMP). By N-terminal or N- and C-terminal fusion of the ice nucleation protein, we successfully displayed the GMP kinase of Lactobacillus bulgaricus and the acetate kinase of E. coli on the surface of E. coli cells. A large amount of soluble target protein was obtained upon induction with 0.2 mM IPTG at 25 °C for 30 h. The conversion of dGMP was up to 91% when catalysed by the surface-displayed enzymes at 37 °C for 4 h. Up to 95% of the GMP was converted after 3 h of reaction. The stability of the whole-cell catalyst at 37 °C was very good. The enzyme activity was maintained above 50% after 9 rounds of recovery. Our research showed that only one-twentieth of the initial substrate concentration of added ATP was sufficient to meet the reaction requirements.

Activation of guanine-ß-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints.

The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

A novel assay of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity in cultured cells and its use for evaluation of cadmium(II) inhibition of this activity.

8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of oxidative modification of dGTP, thatcan be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to 8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer. To elucidate the involvement of 8-oxo-dGTPases in carcinogenesis, an assay of the 8-oxo-dGTPase activity is required. This paper presents such an assay developed for Chinese hamster ovary (CHO) cells that can be applied to any biological material. It includes: (i) a convenient method for preparing 8-oxo-2'-deoxyguanosine 5'-phosphates; (ii) an HPLC/UV quantification of 8-oxo-dGTP hydrolysis products and (iii) separation of 8-oxo-dGTPase activity from interfering 8-oxo-dGTP phosphatase(s). The 8-oxo-dGTPase activity of CHO cells depends on magnesium, has a pH optimum of 8.5, Km for 8-oxo-dGTP of 9.3 microM, and is inhibited by 8-oxo-dGDP, the product of interfering 8-oxo-dGTP phosphatases. The latter must be removed from the assayed samples by ultrafiltration through 30 kDa cut-off membranes. The method was used to test the inhibition by cadmium ions of the activity of 8-oxo-dGTPase in CHO cells. The cells cultured with 0.3-3 microM cadmium(II) acetate for up to 24 h had their 8-oxo-dGTPase activity suppressed in a Cd(II) concentration-dependent manner, down to 70% of the control value.

Inosine-5'-monophosphate analogues as inhibitors of human IMP cyclohydrolase and cellular growth.

The catalytic mechanism for the enzyme, IMP cyclohydrolase, may involve a reaction intermediate with negative charge in the 2-position of the purine ring (Szabados, E., Hindmarsh, E., Phillips, L., Duggleby, R.G. & Christopherson, R.I. (1994) Biochemistry 33, 14237-14245). Three analogues of IMP have been synthesised where fluorine, chlorine or bromine has been substituted in the 2-position on the purine ring. These analogues with an electronegative substituent may resemble a reaction intermediate for IMP cyclohydrolase; 2-fluoro IMP is a potent inhibitor of the enzyme with a Ki value of 0.19 microM, while 2-chloro IMP has a Ki of 1.9 microM and 2-bromo IMP is not inhibitory. However, IMP cyclohydrolase is not inhibited in human CCRF-CEM leukaemia cells exposed to 2-fluoro inosine although it is toxic to these cells with an IC50 value of 4.9 microM.

Generation and elimination of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate, a mutagenic substrate for DNA synthesis, in human cells.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a potent mutagenic substrate for DNA synthesis. The present study deals with generation and degradation of 8-oxo-dGTP in the nucleotide pool of human cells. (1) 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP by nucleoside diphosphate kinase. (2) 8-Oxo-dGTP is rapidly degraded to 8-oxo-dGMP by cellular 8-oxo-dGTPase activity. 8-Oxo-dGMP thus produced cannot be rephosphorylated; guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding nucleoside diphosphates, is totally inactive for 8-oxo-dGMP. (3) 8-Oxo-dGMP is further degraded to 8-oxo-deoxyguanosine by a nucleotidase. The enzyme was partially purified from an extract of human Jurkat cells, and the mode of action was elucidated. 8-Oxo-dGMP is the most preferred substrate of the enzyme, and other nucleoside monophosphates are cleaved at significantly lower rates: Km for 8-oxo-dGMP is 10 times lower than that for dGMP, the second best substrate for the enzyme. The enzyme appears to convert 8-oxo-dGMP, which accumulates in the cellular nucleotide pool, to a form readily excretable to the cell exterior.

Eicar (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide). A novel potent inhibitor of inosinate dehydrogenase activity and guanylate biosynthesis.

EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide) is a cytostatic agent that inhibits murine leukemia L1210 and human lymphocyte CEM cells at a 50% inhibitory concentration of 0.80-1.4 microM, respectively. EICAR causes a rapid and marked inhibition of inosinate (IMP) dehydrogenase (EC 1.1.1.205) activity in intact L1210 and CEM cells reflected by a concentration-dependent accumulation of IMP and depletion of GTP and dGTP levels. EICAR 5'-monophosphate is a potent inhibitor of purified L1210 cell IMP dehydrogenase (Ki/Km 0.06). Inhibition of IMP dehydrogenase by EICAR 5'-monophosphate is competitive with respect to IMP. L1210 cells that were selected for resistance to the cytostatic action of EICAR proved to be adenosine kinase-deficient. Also, studies with other mutant L1210 and CEM cell lines revealed that adenosine kinase, as well as an alternative pathway, may be responsible for the conversion of EICAR to its 5'-monophosphate. Purified 2'-deoxycytidine kinase, 2'-deoxyguanosine kinase, cytosolic 5'-nucleotidase, and nicotinamide dinucleotide (NAD) pyrophosphorylase do not seem to be markedly involved in the metabolism of EICAR.

Detection in situ of gamma-ray-induced DNA strand breaks in single cells: enzymatic labelling of free 3'-OH ends.

We report a procedure allowing the detection and counting of free 3'-OH DNA strand extremities in single cells in situ. Terminal transferase (TdT) catalysed the incorporation of 3H-dGMP into fixed nuclei of human colonic adenocarcinoma cells (HT29), using free 3'-OH ends as initiator. Radioactivity was detected by autoradiography and determined quantitatively with a rapid image-processing system for grain counting. The initiator activity for TdT increases with the dose of gamma-rays in the dose range 2.5-20 Gy.

B cells as well as T cells form deoxynucleotides from either deoxyadenosine or deoxyguanosine.

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.