Enzyme Target Site Research Articles

Synthesis, Antibacterial, and Molecular Docking Study of Novel 2-Chloro-8-Methoxy-3-Aryl-[1,3] Benzoxazine Derivatives using Vilsmeier Reagent

Reducing of ethyl 4-((2-hydroxy-3-methoxybenzylidene)amino)benzoate (1) afford ethyl 4-((2-hydroxy-3-methoxybenzyl)amino)benzoate (2). Reaction of this compound with Vilsmeier reagent affords novel 2-chloro-[1,3] benzoxazine ring (3). The corresponding acid hydrazide of compound 3 was synthesized from reaction of compound (3) with hydrazine hydrate. Newly series of hydrazones (5a–i) were synthesized from reaction of acid hydrazide with various aryl aldehydes. Antibacterial activity of the hydrazones was secerned utilizing gram-negative and gram-positive bacteria. Compound (5b) and (5c) exhibited significant antibacterial ability against both gram-negative and gram-positive bacteria, while the compounds (5a) showed mild antibacterial activity. Compounds (5d–i) did not display notable activity. The molecular docking of synthesised compounds were tested inside the pocket of bacterial gyrase enzyme target site by using MOE 2015 software, which acts as Adenosine triphosphate (ATP)-binding domain bacterial gyrase enzyme pocket and novobiocin was used as reference.

Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure.

The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5′TC3′ or 5′CT3′ dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

AbstractQuinoa (Chenopodium quinoaWilld) is a dicotyledonous annual species belonging to the family Amaranthaceae, which is nutritionally well balanced in terms of its oil, protein and carbohydrate content. Targeting-induced local lesions in genomes (the TILLING strategy) was employed to find mutations in acetolactate synthase (AHAS) genes in a mutant quinoa population. TheAHASgenes were targeted because they are common enzyme target sites for five herbicide groups. Ethyl methane sulfonate (EMS) was used to induce mutations in theAHASgenes; it was found that 2% EMS allowed a mutation frequency of one mutation every 203 kilobases to be established. In the mutant population created, a screening strategy using pre-selection phenotypic data and next-generation sequencing (NGS) allowed identification of a mutation that alters the amino acid composition of this species (nucleotide 1231 codon GTT→ATT, Val→Ile); however, this mutation did not result in herbicide resistance. The current work shows that TILLING combined with the high-throughput of NGS technologies and an overlapping pool design provides an efficient and economical method for detecting induced mutations in pools of individuals.

One-pot synthesis of anilides, herbicidal activity and molecular docking study.

In the context of the demand for more efficient herbicides, the aim of the present work was to synthesize anilides via simple methods, and evaluate their herbicidal activities through seed germination assays. In silico studies were carried out to identify the enzyme target sites in plants for the most active anilides. A total of 18 anilides were prepared via one-pot reaction in yields that varied from 36 to 98% through reactions of anilines with sorbic chloride and hexanoic anhydride. According to seed germination assays in three dicotyledonous and one monocotyledonous plant species, the most active anilides showed root and shoot growth inhibition superior to that of Dual (S-metolachlor). In silico studies indicated that histone deacetylase was the probable enzyme target site in plants for these substances. The affinities of the most active anilides for the binding sites of this enzyme were equal to or higher than those calculated for its inhibitors. Anilides 4d, 4e, 4 g, and 4 h are promising candidates for the development of novel herbicides. According to in silico studies, they inhibit histone deacetylase in plants, which can be exploited for the development of new weed control methods. © 2018 Society of Chemical Industry.

Characterization of glyphosate resistance in cloned Amaranthus palmeri plants

Glyphosate‐resistant Palmer amaranth from Georgia (GA), USA, possesses multiple copies of the gene that encodes 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), the enzyme target site of this herbicide. The cloned plants of glyphosate‐resistant and glyphosate‐susceptible Palmer amaranth biotypes from Mississippi (MS), USA, and GA were evaluated for glyphosate injury (digital imaging) in leaf disc bioassays. Four groups (three resistant groups: two from MS [G and R] and one from GA [C7]; one susceptible group from GA [C3]) were chosen for cloning to facilitate long‐term studies. After exposure to glyphosate (1.0 mmol L–1, 144 h), the level of injury (mean value) was low in the resistant groups, while a higher level of injury was found in the susceptible group. However, the individual injury values within all groups varied widely. The mean EPSPS gene copy number of these groups was G ≥ R > C7 >>> C3. However, a higher copy number did not always convey increased resistance in these bioassays. When the copy number was high (>20), 81.5% of the bioassayed plants exhibited little or no injury and only ∼20% were significantly injured, while 50% of the plants with a low copy number (<20) remained healthy. Overall, no strong statistical correlation of the copy number versus injury occurred in these cloned plants and no statistical relationship of resistance and copy number with the sex of the MS plants was observed. The results suggest that although an elevated copy number of the EPSPS gene can instill resistance, other mechanisms might contribute to the overall glyphosate resistance of Palmer amaranth in these plants.

C.U.R.R.F. (Codon Usage regarding Restriction Finder): A Free Java®-Based Tool to Detect Potential Restriction Sites in Both Coding and Non-Coding DNA Sequences

The synthesis of complete genes is becoming a more and more popular approach in heterologous gene expression. Reasons for this are the decreasing prices and the numerous advantages in comparison to classic molecular cloning methods. Two of these advantages are the possibility to adapt the codon usage to the host organism and the option to introduce restriction enzyme target sites of choice. C.U.R.R.F. (Codon Usage regarding Restriction Finder) is a free Java(®)-based software program which is able to detect possible restriction sites in both coding and non-coding DNA sequences by introducing multiple silent or non-silent mutations, respectively. The deviation of an alternative sequence containing a desired restriction motive from the sequence with the optimal codon usage is considered during the search of potential restriction sites in coding DNA and mRNA sequences as well as protein sequences. C.U.R.R.F is available at http://www.zvm.tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/mikrobiologie/allgemeine_mikrobiologie/currf.

Simultaneous Single‐Molecule Mapping of Protein‐DNA Interactions and DNA Methylation by MAPit

Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein-DNA interactions by probing with cytosine-modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite-converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC-methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed.

Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity.

LOVely enzymes – towards engineering light‐controllable biocatalysts

SummaryLight control over enzyme function represents a novel and exciting field of biocatalysis research. Blue‐light photoreceptors of the Light, Oxygen, Voltage (LOV) family have recently been investigated for their applicability as photoactive switches. We discuss here the primary photochemical events leading to light activation of LOV domains as well as the proposed signal propagation mechanism to the respective effector domain. Furthermore, we describe the construction of LOV fusions to different effector domains, namely a dihydrofolate reductase from Escherichia coli and a lipase from Bacillus subtilis. Both fusion partners retained functionality, and alteration of enzyme activity by light was also demonstrated. Hence, it appears that fusion of LOV photoreceptors to functional enzyme target sites via appropriate linker structures may represent a straightforward strategy to design light controllable biocatalysts.

Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation.

Intrinsic nucleosome dynamics termed "site exposure" provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on nucleosome translocation, the other on dynamic nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside nucleosomes when extra DNA (onto which the nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural nucleosome positioning sequence, the other on the well-studied 5S rRNA gene nucleosome positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from nucleosome translocation. We conclude that site exposure in nucleosomes in vitro occurs via a nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.

Interaction of Cu(II) and Cu(II)-anthracycline complexes with protein kinase C. Spectromagnetic assessment of the inhibitory effect

Anthracycline antitumor drugs weakly inhibit protein kinase C (PKC) activity; this effect is enhanced by coordination with transition metal ions. The present study shows that copper(II) by itself inhibits PKC activity; when Cu(II) is present as the anthracycline-Cu(II) complex [Cu(EpiDXR) 2], a significant enhancement is observed as compared to the inhibitory effects of either metal or drug alone. Both regulatory and catalytic sites of PKC are involved in this effect. Electron paramagnetic resonance (EPR) studies indicate an interaction between PKC and Cu(II). It is suggested that Cu(II) is responsible for the inhibitory action of Cu(II)-anthracycline complexes on PKC; anthracycline molecules potentiate this effect, possibly by stabilizing Cu(II) and increasing its availability at enzyme target sites.

Structural features of transposed human VKgenes and implications for the mechanism of their transpositions

The genes encoding the variable, joining and constant regions of human immunoglobulin light chains have been localized to the short arm of chromosome 2. However, several VK genes lie outside of the locus: a single copy cluster of five VK genes is located on chromosome 22; an isolated but amplified VkI gene is found on chromosome 1; and several isolated VkI genes are on as-yet-unidentified chromosomes other than chromosome 2. Vk genes not contained within the kappa locus are termed orphons. We have attempted to gain insight into the mechanism of transposition of both the chromosome 22 cluster and the several amplified VkI genes by searching in the kappa locus for a parent copy of the former, and by analyzing the junctions between transposed VKI-containing segments and adjacent non-amplified regions. The chromosome 22 orphon cluster must have been non-duplicatively transposed. Sequence features at the junctions of this and other orphon regions are direct and inverted repeats, and, in one case, an Alu repeat. These unusual features may have predisposed the orphon regions to transposition by serving as target sites for enzymes involved in recombination.

Tentoxin, a Chlorosis-Inducing Toxin fromAlternariaas a Potential Herbicide

The potential for use of compounds isolated from diverse sources, ranging from microbial to higher plants, has received increasing attention. These natural products represent a vast array of chemistries and mechanisms of action often having the requisite specificities to be ideal herbicides. Tentoxin, a cyclic tetrapeptide, has potent chlorosis activity on a variety of weeds of soybean and corn while not affecting these crops. Investigations into this toxin's mechanism suggest several enzyme target sites for the development of herbicidal analogues, each having the inherent specificity of the parent compound. Structure/activity relationships determined so far may permit a directed synthesis of efficacious analogues. New insights into biosynthesis of the toxin suggest possible means of increasing production to allow economical exploitation of the toxin itself as a herbicide.

Studies on the mode of action of asulam in bracken (Pteridium aquilinumL. Kuhn)

SynopsisThe efficiency of bracken control using foliage-applied translocated herbicides depends upon the accumulation of a potentially lethal concentration of the active ingredient at the sites of action, the rhizome buds. There is evidence that the level of herbicide accumulation at these target sites is determined by a number of factors including the efficiency of cuticle penetration, absorption, translocation, metabolism and activity at the enzyme target sites. The level of metabolic activity of the rhizome/frond buds may be critical, dormant buds possibly receiving a sublethal dose. These buds may later become active and act as foci for reinfestation.The nature of these problems is discussed with particular reference to studies carried out on the mode of action of [14C]asulam.