365-07-1Relevant academic research and scientific papers
Characterization of Lhr-Core DNA helicase and manganese-dependent DNA nuclease components of a bacterial gene cluster encoding nucleic acid repair enzymes
Ejaz, Anam,Shuman, Stewart
, p. 17491 - 17504 (2018/11/23)
Lhr is a large superfamily 2 helicase present in mycobacteria and a moderate range of other bacterial taxa. A shorter version of Lhr, here referred to as Lhr-Core, is distributed widely in bacteria, where it is often encoded in a gene cluster along with predicted binuclear metallo-phosphoesterase (MPE), ATP-dependent DNA ligase, and metallo-β-lactamase exonuclease enzymes. Here we characterized the Lhr-Core and MPE proteins from Pseudomonas putida. We report that P. putida Lhr-Core is an ssDNA-dependent ATPase/dATPase (Km, 0.37 mM ATP; kcat, 3.3 s–1), an ATP-dependent 3'-to-5' single-stranded DNA translocase, and an ATP-dependent 3'-to-5' helicase. Lhr-Core unwinds 3'-tailed duplexes in which the loading/tracking strand is DNA and the displaced strand is either DNA or RNA. We found that P. putida MPE is a manganese-dependent phosphodiesterase that releases p-nitrophenol from bis-p-nitrophenyl phosphate (kcat, 212 s–1) and p-nitrophenyl-5'-thymidylate (kcat, 34 s-1) but displays no detectable phosphomonoesterase activity against p-nitrophenyl phosphate. MPE is also a manganese-dependent DNA endonuclease that sequentially converts a closed-circle plasmid DNA to nicked circle and linear forms prior to degrading the linear DNA to produce progressively smaller fragments. The biochemical activities of MPE and a structure predicted in Phyre2 point to MPE as a new bacterial homolog of Mre11. Genetic linkage of a helicase and DNA nuclease with a ligase and a putative exonuclease (a predicted homolog of the SNM1/Apollo family of nucleases) suggests that these enzymes comprise or participate in a bacterial DNA repair pathway.
Enzymatic Production of Non-Natural Nucleoside-5′-Monophosphates by a Thermostable Uracil Phosphoribosyltransferase
del Arco, Jon,Acosta, Javier,Pereira, Humberto M.,Perona, Almudena,Lokanath, Neratur K.,Kunishima, Naoki,Fernández-Lucas, Jesús
, p. 439 - 448 (2017/12/13)
The use of enzymes as biocatalysts applied to synthesis of modified nucleoside-5′-monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo-, regio-, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate-binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several 5 and 6-substituted pyrimidines as substrates. These experimental results suggest TtUPRT could be a valuable biocatalyst for the synthesis of modified NMPs.
Enzyme Activation with a Synthetic Catalytic Co-enzyme Intermediate: Nucleotide Methylation by Flavoenzymes
Bou-Nader, Charles,Cornu, David,Guerineau, Vincent,Fogeron, Thibault,Fontecave, Marc,Hamdane, Djemel
supporting information, p. 12523 - 12527 (2017/09/12)
To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.
New phosphorylating reagents for deoxyribonucleosides and oligonucleotides
Romanucci, Valeria,Zarrelli, Armando,Guaragna, Annalisa,Di Marino, Cinzia,Di Fabio, Giovanni
supporting information, p. 1227 - 1229 (2017/03/02)
New phosphorylating reagents 1 and 2 were prepared in three steps from 4-hydroxybenzaldehyde. They showed good efficiency in the solid phase synthesis of 5′-phosphate monoester nucleosides. End-phosphate DNA sequence synthesis demonstrated the efficiency of the new reagents (1 and 2) according to the general procedure of automated DNA synthesis. The oxidation of P(III) to P(V) and the removal of benzyl protecting groups were achieved in a single step by treatment with a 0.02 M I2/pyridine/H2O solution. Due to this one-pot treatment, it is possible to use the phosphorylating reagents (1 and 2) for the synthesis of base-sensitive ODNs. The reagents 1 and 2 are unique among phosphorylating reagents.
Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-hydroxy-dCMP
Ludwiczak, Jan,Maj, Piotr,Wilk, Piotr,Fraczyk, Tomasz,Ruman, Tomasz,Kierdaszuk, Borys,Jarmu?a, Adam,Rode, Wojciech
, p. 1333 - 1341 (2016/04/09)
Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy-dCMP was found to be strongly dependent on [Mg2+], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells.
Synthesis and application of isotopically labeled flavin nucleotides
Mishanina, Tatiana V.,Kohen, Amnon
, p. 370 - 375 (2015/08/06)
Flavin nucleotides, i.e. flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are utilized as prosthetic groups and/or substrates by a myriad of proteins, ranging from metabolic enzymes to light receptors. Isotopically labeled flavins have served as invaluable tools in probing the structure and function of these flavoproteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides from commercially available labeled riboflavin and ATP. The synthetic procedure employs a bifunctional enzyme, Corynebacterium ammoniagenes FAD synthetase, that sequentially converts riboflavin to FMN and then to FAD. The final flavin product (FMN or FAD) is controlled by the concentration of ATP in the reaction. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors. Flavin nucleotides are utilized as prosthetic groups and/or substrates by a myriad of proteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors.
Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
, p. 1575 - 1581 (2015/02/19)
The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides
Serra, Immacolata,Conti, Silvia,Piskur, Jure,Clausen, Anders R.,Munch-Petersen, Birgitte,Terreni, Marco,Ubiali, Daniela
, p. 563 - 570 (2014/05/20)
Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).
Concerted versus stepwise mechanism in thymidylate synthase
Islam, Zahidul,Strutzenberg, Timothy S.,Gurevic, Ilya,Kohen, Amnon
supporting information, p. 9850 - 9853 (2014/08/05)
Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.
Synthesis of nucleoside 5′-tetraphosphates containing terminal fluorescent labels via activated cyclic trimetaphosphate
Mohamady, Samy,Taylor, Scott D.
, p. 2308 - 2313 (2014/04/03)
2′-Deoxynucleotide 5′-tetraphosphates in which a fluorescent label is attached to the terminal phosphate are used as key reagents in high-throughput DNA sequencing techniques and in single nucleotide polymorphism typing assays. We demonstrate that this class of compounds can be prepared by reacting fluorophores such as 7-hydroxy-4-methylcoumarin, methylfluorescein, fluorescein and resorufin with an activated form of cyclic trimetaphosphate to give intermediate 11. Reaction of 11 with 2′-deoxynucleoside 5′-monophosphates or a nucleoside 5′-monophosphate gave the target compounds in good yield.
