Terminal Phosphate Moiety Research Articles

YvcI from Bacillus subtilis has in vitro RNA pyrophosphohydrolase activity

RNA degradation is one of several ways for organisms to regulate gene expression. In bacteria, the removal of two terminal phosphate moieties as orthophosphate (Bacillus subtilis) or pyrophosphate (Escherichia coli) triggers ribonucleolytic decay of primary transcripts by 5'-monophosphate-dependent ribonucleases. In the soil-dwelling firmicute species B. subtilis, the RNA pyrophosphohydrolase BsRppH, a member of the Nudix family, triggers RNA turnover by converting primary transcripts to 5'-monophospate RNA. In addition to BsRppH, a source of redundant activity in B. subtilis has been proposed. Here, using recombinant protein expression and in vitro enzyme assays, we provide evidence for several additional RNA pyrophosphohydrolases, among them MutT, NudF, YmaB, and YvcI in B. subtilis We found that in vitro, YvcI converts RNA 5'-di- and triphosphates into monophosphates in the presence of manganese at neutral to slightly acidic pH. It preferred G-initiating RNAs and required at least one unpaired nucleotide at the 5'-end of its substrates, with the 5'-terminal nucleotide determining whether primarily ortho- or pyrophosphate is released. Exchanges of catalytically important glutamate residues in the Nudix motif impaired or abolished the enzymatic activity of YvcI. In summary, the results of our extensive in vitro biochemical characterization raise the possibility that YvcI is an additional RNA pyrophosphohydrolase in B. subtilis.

A continuous fluorescence assay for the characterization of Nudix hydrolases

The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske–SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α–β position, MutT cleaves at the β–γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α–β position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.

Synthesis and anticancer activity of a series of norcantharidin analogues

Cantharidin (1) and norcantharidin (2) display high levels of anticancer activity against a broad range of tumour cell lines. Synthetic manipulation of norcantharidin yields (3S,3aR,4S,7R,7aS)-3-hydroxyhexahydro-4,7-epoxyisobenzofuran-1(3H)-one (3), which also displays a high level of anticancer activity against tumour cells but interestingly, shows selectivity towards HT29 (colon; GI50 = 14 μM) and SJ-G2 (glioblastoma; GI50 = 15 μM) cell lines. Substitution at the hydroxyl group of the cyclic lactone within (3) produces a diasteromeric pair of products that have no difference in cytotoxicity over the cell lines tested. Incorporation of an isopropyl tail at this position (16) produced the most promising compound of this series to date, with strong selectivity towards HT29 (colon; GI50 = 19 μM) and SJ-G2 (glioblastoma; GI50 = 21 μM) cell lines but completely void of any activity against the remaining tumour cell lines (GI50 > 100 μM), as per the parent molecule. We also discovered that the introduction of a terminal phosphate moiety (28) at the same position produced a different trend in cytotoxicity with strong activity in BE2-C (neuroblastoma; GI50 = 9 μM) cells; suggestive of an alternate mode of action.

Real-time DNA Synthesis Dynamics Of Single f29 DNA Polymerase Molecules With Base Pair Resolution

Real-time observation of uninterrupted DNA synthesis by individual f29 DNA polymerase molecules is presented. Four spectrally distinct fluorophores were used to label the four nucleotides via the terminal phosphate moiety, enabling identification of nucleotide incorporation events into the growing DNA strand with base-pair resolution, and correlating these events to the DNA template sequence. Immobilization of polymerase molecules inside zero-mode waveguide nanostructures allowed detection of incorporation events at 100-500 nM nucleotide concentrations, resulting in polymerization rates of ∼1-5 bases/s, and lasting for thousands of bases synthesized. Many aspects of the underlying DNA polymerase dynamics are directly observable, such as nucleotide binding, catalysis, pausing, persistence of distinct kinetic states, DNA template sequence context effects, and switching between polymerization and exonuclease activities.

Real-Time DNA Sequencing from Single Polymerase Molecules

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

Surface complexation of condensed phosphate to aluminum hydroxide: An ATR-FTIR spectroscopic investigation

Two model compounds, sodium pyrophosphate (pyro-P) and sodium tripolyphosphate (tripoly-P), were employed to elucidate the binding mechanisms of condensed phosphate on aluminum hydroxide by utilizing attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. Peak assignments for the condensed phosphates in the solution phase and those adsorbed on the surface of aluminum hydroxide were made. Electron delocalization and polarization were employed to explain the peak shifts and the complexation of condensed phosphate with aluminum hydroxide. The tripoly-P and pyro-P were adsorbed on aluminum hydroxide by forming inner-sphere complexes. The adsorbed condensed phosphates were deprotonated in the pH range from 4 to 10. Monodentate, bidentate, and binuclear complexes were formed when pyro-P was adsorbed on aluminum hydroxide, while monodentate and binuclear complexes were formed when tripoly-P was adsorbed. Based on the FTIR data, we proposed that when either bidentate or binuclear complexes were formed, the two oxygen atoms participating in the complexation with aluminum hydroxide could not be originated from the same terminal phosphate moiety. The Al O bond formed in the complexation of pyro-P or tripoly-P with aluminum hydroxide ( AlPO − 3) was not as strong as the H O bond in terminal HPO − 3. The bridging PO − 2 of tripoly-P did not coordinate with aluminum hydroxide. The real-time ATR-FTIR study on condensed phosphate adsorption revealed that a long contact time between condensed phosphates and aluminum hydroxide particles can result in a transformation of an initially formed species into a thermodynamically more stable phase.

Investigation of Oligonucleotide Fragmentation with Matrix-assisted Laser Desorption/Ionization Fourier-transform Mass Spectrometry and Sustained Off-resonance Irradiation

Matrix-assisted laser desorption/ionization (MALDI) can be combined with Fourier-transform ion cyclotron resonance mass spectrometry (FTMS) for the detailed structural examination of biomolecules such as peptides and oligonucleotides. We have been able to detect molecular ions for bovine heart cytochrome c (MW = 12,327) by MALDI-FTMS (355 nm laser desorption, 2,5-dihydroxybenzoic acid matrix). Although the mass resolution of these molecular ions is poor, the experiments verify that the MALDI-FTMS mass range for our 3-tesla instrument is in excess of m/z 12,000. Accurate mass measurements and selective dissociation experiments were used to examine the fragmentation pathways of small oligonucleotides in detail. Sustained off-resonance irradiation (SORI) was found to be superior to conventional on-resonance collisionally activated dissociation (CAD) for the efficient dissociation and detection of fragment ions for oligonucleotides. These experiments indicated that oligonucleotide fragmentation is a complex process and results not only from simple elimination of nucleic bases and cleavages of phosphate ester bonds, but also by rearrangement processes in which a terminal phosphate moiety can be transferred to an internal phosphate group.

42] Criteria for the classification of protein kinases

Protein kinases catalyze the transfer of the terminal phosphate moiety of ATP to a variety of different protein substrates. Initially the classification of these enzymes was, in accord with standard enzymological nomenclature, based on specificity with respect to the protein substrate. More recently it has become recognized that cyclic AMP (cAMP) regulates the activity of some, but not all, of these enzymes, and from experimentation to date it would appear that many cAMP-regulated protein kinases exhibit broad protein substrate specificity. Thus, classical nomenclature becomes inoperable at the experimental level, although in the future cAMP-regnlated protein kinases of unique protein substrate specificity may be recognized. The activation of cAMP-regulated protein kinases occurs by a cyclic nucleotide-promoted dissociation of the holoenzyme (designated RC) to yield the active catalytic species (C) and a complex consisting of regulatory subunit (R) and cAMP.