Kinetic Analysis of Prodrug Activation and ATP/UTP Substrate Preference of Nine Human Deoxycytidine Kinase Mutants

Deoxynucleoside analogues are prodrugs that canfunction as inhibitors of both viral and cellular DNA replication processes. They are important in anti-cancer therapy because they hinder DNA synthesis and cellular mitosis. Within the cell, deoxyribonucleotides are synthesized using the salvage pathways by converting the unphosphorylated nucleosides to their mono, di- and tri-phosphate forms using a phosphoryl donor: ATP or UTP. Human deoxycytidine kinase (dCK) is the first and rate-limiting enzyme in this process. The dCK protein uses nucleotide triphosphates to phosphorylate several clinically important nucleoside analogue prodrugs in addition to its natural substrates. The preferred physiological phosphoryl donor for dCK is UTP although it is less prevalent in the human body than ATP. Our objective is to improve the understanding of the phosphate-donor binding loop of dCK by kinetic analysis of a series of mutants of Asp241 and Phe242. These mutants were designed in an attempt to improve the activity of dCK with phosphate donors. Results show several mutants with improved kinetics and some with an ATP donor preference over UTP

Development of a Novel GC/MS Method for the Detection of Nicotinamide and Activity of ADP-Ribosylating Toxins

ADP-ribosylating toxin enzymes break NADH (Figure1) and transfer the ADP-ribosyl group to a residue on a target protein, permanently inactivating or denaturing the protein. This activity is typically detected with a radioassay, which is expensive and requires radioactive materials. ADP-ribosylation corresponds with the release of nicotinamide. It is possible to detect nicotinamide with a Gas Chromatograph/Mass Spectrometer (GC/MS) (Jacobson, Dame, Pyrek & Jacobson, 1995). The purpose of this study is to measure ADP-ribosylation activity using GC/MS by detecting the liberated nicotinamide. By derivatizing the nicotinamide, the detection limit was lowered to 0.5ng/μl. Control measurements of ADP-ribosylation activity by a cholera toxin protein found low levels of nicotinamide contamination

Structural Biology of Bacterial Multidrug Resistance Gene Regulators

Multidrug resistance (mdr)1 can be defined broadly as the ability of a cell to survive ordinarily lethal doses of more than one drug. Clearly, such resistance is a critical problem in the treatment of fungal and bacterial infections and cancer. Four general, but nonexclusive, mechanisms give rise to multidrug resistance: 1) detoxification by enzymatic modification or cleavage of drug; 2) genetic alteration of the intra- or extracellular targets; 3) decreased permeability of the cell membrane; and 4) active drug extrusion by multidrug transporters. Paramount to our understanding of mdr is the issue of recognition of structurally dissimilar substrates and how drug binding effects function. In bacteria many multidrug transporters are regulated directly (locally) by transcription factors, which also bind the substrates of these transporters, i.e. the drug can act as a transcriptional coactivator or inducer. Multiple mdr transporter genes are also regulated globally by activators such as MarA that do not necessarily bind drugs (1). The regulators are of keen interest because they are more amenable to structural studies than the membrane-bound transporters and thus offer a greater chance to obtain high resolution views of multidrug binding. Moreover, the local gene regulators are equally interesting as their DNA complexes directly reveal the mechanism of mdrtransporter gene regulation. This minireview will summarize the structures of known bacterial mdr regulators. Because our focus is more structural the reader is referred to one of several recent reviews that discuss the more biological aspects of global and local mdr regulation (2-5)

Crystal Structure of MtaN, a Global Multidrug Transporter Gene Activator

MtaN (Multidrug Transporter Activation, N terminus) is a constitutive, transcriptionally active 109-residue truncation mutant, which contains only the N-terminal DNA-binding and dimerization domains of MerR family member Mta. The 2.75 Å resolution crystal structure of apo-MtaN reveals a winged helix-turn-helix protein with a protruding 8-turn helix (α5) that is involved in dimerization by the formation of an antiparallel coiled-coil. The hydrophobic core and helices α1 through α4 are structurally homologous to MerR family member BmrR bound to DNA, whereas one wing (Wing 1) is shifted. Differences between the orientation of α5 with respect to the core and the revolution of the antiparallel coiled-coil lead to significantly altered conformations of MtaN and BmrR dimers. These shifts result in a conformation of MtaN that appears to be incompatible with the transcription activation mechanism of BmrR and suggest that additional DNA-induced structural changes are necessary

River ecosystem conceptual models and non‐perennial rivers: A critical review

Conceptual models underpin river ecosystem research. However, current models focus on continuously flowing rivers and few explicitly address characteristics such as flow cessation and drying. The applicability of existing conceptual models to nonperennial rivers that cease to flow (intermittent rivers and ephemeral streams, IRES) has not been evaluated. We reviewed 18 models, finding that they collectively describe main drivers of biogeochemical and ecological patterns and processes longitudinally (upstream-downstream), laterally (channel-riparian-floodplain), vertically (surface water-groundwater), and temporally across local and landscape scales. However, perennial rivers are longitudinally continuous while IRES are longitudinally discontinuous. Whereas perennial rivers have bidirectional lateral connections between aquatic and terrestrial ecosystems, in IRES, this connection is unidirectional for much of the time, from terrestrial-to-aquatic only. Vertical connectivity between surface and subsurface water occurs bidirectionally and is temporally consistent in perennial rivers. However, in IRES, this exchange is temporally variable, and can become unidirectional during drying or rewetting phases. Finally, drying adds another dimension of flow variation to be considered across temporal and spatial scales in IRES, much as flooding is considered as a temporally and spatially dynamic process in perennial rivers. Here, we focus on ways in which existing models could be modified to accommodate drying as a fundamental process that can alter these patterns and processes across spatial and temporal dimensions in streams. This perspective is needed to support river science and management in our era of rapid global change, including increasing duration, frequency, and occurrence of drying.info:eu-repo/semantics/publishedVersio

Kinetic Analysis of Prodrug Activation and ATP/UTP Substrate Preference of Nine Human Deoxycytidine Kinase Mutants

Deoxynucleoside analogues are prodrugs that canfunction as inhibitors of both viral and cellular DNA replication processes. They are important in anti-cancer therapy because they hinder DNA synthesis and cellular mitosis. Within the cell, deoxyribonucleotides are synthesized using the salvage pathways by converting the unphosphorylated nucleosides to their mono, di- and tri-phosphate forms using a phosphoryl donor: ATP or UTP. Human deoxycytidine kinase (dCK) is the first and rate-limiting enzyme in this process. The dCK protein uses nucleotide triphosphates to phosphorylate several clinically important nucleoside analogue prodrugs in addition to its natural substrates. The preferred physiological phosphoryl donor for dCK is UTP although it is less prevalent in the human body than ATP. Our objective is to improve the understanding of the phosphate-donor binding loop of dCK by kinetic analysis of a series of mutants of Asp241 and Phe242. These mutants were designed in an attempt to improve the activity of dCK with phosphate donors. Results show several mutants with improved kinetics and some with an ATP donor preference over UTP

Progress Toward Removal of the Purification Tag in a Putative Toxin Protein via Site-Specific Mutagenesis and Proteolytic Cleavage

Putative toxin protein BC_2332, isolated from Bacillus cereus, which is a relative of Bacillus anthracis, shows similarity in the active site to ADP-ribosylating proteins present in cholera and diphtheria. Preliminary attempts to measure the activity of the putative toxin were unsuccessful leading to the hypothesis that the purification tag is hindering the activity. This hypothesis is supported by crystal structure analysis showing the putative active site occupied by the purification tag. Our objective was to remove the purification tag, via two different methods, in order to relieve potential inhibition. Both methods have faced obstacles and the work is continuing

Development of a Novel GC/MS Method for the Detection of Nicotinamide and Activity of ADP-Ribosylating Toxins

ADP-ribosylating toxin enzymes break NADH (Figure1) and transfer the ADP-ribosyl group to a residue on a target protein, permanently inactivating or denaturing the protein. This activity is typically detected with a radioassay, which is expensive and requires radioactive materials. ADP-ribosylation corresponds with the release of nicotinamide. It is possible to detect nicotinamide with a Gas Chromatograph/Mass Spectrometer (GC/MS) (Jacobson, Dame, Pyrek & Jacobson, 1995). The purpose of this study is to measure ADP-ribosylation activity using GC/MS by detecting the liberated nicotinamide. By derivatizing the nicotinamide, the detection limit was lowered to 0.5ng/μl. Control measurements of ADP-ribosylation activity by a cholera toxin protein found low levels of nicotinamide contamination

Structural Basis for the Preference of UTP over ATP in Human Deoxycytidine Kinase: Illuminating the Role of Main-Chain Reorganization

Human deoxycytidine kinase (dCK) uses nucleoside triphosphates to phosphorylate several clinically important prodrugs in addition to its natural substrates. Although UTP is the preferred phosphoryl donor for this reaction, our previous studies reported dCK structures solely containing ADP in the phosphoryl donor binding site. To determine the molecular basis of the kinetically observed phosphoryl donor preference, we solved crystal structures of a dCK variant lacking a flexible insert (residues 65−79) but having similar catalytic properties as wild type, in complex with deoxycytidine (dC) and UDP, and in the presence of dC but the absence of UDP or ADP. These structures reveal major changes in the donor base binding loop (residues 240−247) between the UDP-bound and ADP-bound forms, involving significant main-chain rearrangement. This loop is disordered in the dCK-dC structure, which lacks a ligand at the phosphoryl donor site. In comparison with the ADP-bound form, in the presence of UDP this loop is shifted inward to make closer contact to the smaller uracil base. These structures illuminate the phosphoryl donor binding and preference mechanisms of dCK