45 research outputs found

    The Role of Electrostatic Interactions in Complex Formation between Bacterial Luciferase and NADPH:FMN-oxidoreductase

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    A possible mechanism of complex formation between bacterial luciferase and NADPH:FMNoxidoreductase from Vibrio harveyi sustained by electrostatic forces is studied. The complex between the enzymes is important for a direct FMNH2 transfer without a contact with solvent, which could cause a rapid autooxidation and the formation of reactive oxygen species. In the current work the diversity of possible relative positions of NADPH:FMN-oxidoreductase and luciferase was obtained with Monte-Carlo sampling governed by oxidoreductase internal charged groups and electrostatic field caused by luciferase. Among the structures with the minimal energies, the one was found that has a proper active sites orientation for a direct FMNH2 transfer. Possible role of hydrogen bonding between Arg291 and Gln197 of luciferase and oxidoreductase, respectively, in stabilization of this complex is propose

    Applications of luminous bacteria enzymes in toxicology

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    This review describes the principle and applications of bioluminescent enzymatic toxicity bioassays. This type of assays uses bacterial coupled enzyme systems: NADH:FMN-oxidoreductase and luciferase to replace living organisms in developing cost-competitive biosensors for environmental, medical and industrial applications. These biosensors instantly signal chemical and biological hazards and allow for detecting a great amount of toxic compounds with advantages associated with fast results, high sensitivity, simplicity, low cost and safety of the procedure

    Functional divergence between evolutionary related LuxG and Fre oxidoreductases of luminous bacteria

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    In luminous bacteria NAD(P)H:flavin-oxidoreductases LuxG and Fre there are homologous enzymes that could provide a luciferase with reduced flavin. While Fre functions as a housekeeping enzyme, LuxG appears to be a source of reduced flavin for bioluminescence as it is transcribed together with luciferase. This study is aimed at providing the basic conception of Fre and LuxG evolution and revealing the peculiarities of the active site structure resulted from a functional variation within the oxidoreductase family. A phylogenetic analysis has demonstrated that Fre and LuxG oxidoreductases have evolved separately after the gene duplication event, and consequently, they have acquired changes in the conservation of functionally related sites. Namely, different evolutionary rates have been observed at the site responsible for specificity to flavin substrate (Arg 46). Also Tyr 72 forming a part of a mobile loop involved into FAD binding has been found to be conserved among Fre in contrast to LuxG oxidoreductases. The conservation of different amino acid types in NAD(P)H binding site has been defined for Fre (arginine) and LuxG (proline) oxidoreductases

    Applications of luminous bacteria enzymes in toxicology

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    This review describes the principle and applications of bioluminescent enzymatic toxicity bioassays. This type of assays uses bacterial coupled enzyme systems: NADH:FMN-oxidoreductase and luciferase to replace living organisms in developing cost-competitive biosensors for environmental, medical and industrial applications. These biosensors instantly signal chemical and biological hazards and allow for detecting a great amount of toxic compounds with advantages associated with fast results, high sensitivity, simplicity, low cost and safety of the procedure

    The Role of Electrostatic Interactions in Complex Formation between Bacterial Luciferase and NADPH:FMN-oxidoreductase

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    ΠœΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ молСкулярного Π΄ΠΎΠΊΠΈΠ½Π³Π° исслСдован Π²ΠΎΠ·ΠΌΠΎΠΆΠ½Ρ‹ΠΉ ΠΌΠ΅Ρ…Π°Π½ΠΈΠ·ΠΌ формирования Π±Π΅Π»ΠΊΠΎΠ²ΠΎΠ³ΠΎ комплСкса ΠΌΠ΅ΠΆΠ΄Ρƒ Π±Π°ΠΊΡ‚Π΅Ρ€ΠΈΠ°Π»ΡŒΠ½ΠΎΠΉ Π»ΡŽΡ†ΠΈΡ„Π΅Ρ€Π°Π·ΠΎΠΉ ΠΈ NADPH:FMN-оксидорСдуктазой ΠΈΠ· Vibrio harveyi Π·Π° счСт элСктростатичСских взаимодСйствий. КомплСкс ΠΌΠ΅ΠΆΠ΄Ρƒ исслСдуСмыми Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π°ΠΌΠΈ Π½Π΅ΠΎΠ±Ρ…ΠΎΠ΄ΠΈΠΌ для ΠΏΠ΅Ρ€Π΅Π΄Π°Ρ‡ΠΈ Π»ΡŽΡ†ΠΈΡ„Π΅Ρ€Π°Π·Π΅ субстрата – ΠΌΠΎΠ»Π΅ΠΊΡƒΠ»Ρ‹ FMNH2, которая ΠΏΠΎΠ΄Π²Π΅Ρ€ΠΆΠ΅Π½Π° быстрому Π°Π²Ρ‚ΠΎΠΎΠΊΠΈΡΠ»Π΅Π½ΠΈΡŽ с ΠΎΠ±Ρ€Π°Π·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ Π°ΠΊΡ‚ΠΈΠ²Π½Ρ‹Ρ… Ρ„ΠΎΡ€ΠΌ кислорода ΠΏΡ€ΠΈ ΠΊΠΎΠ½Ρ‚Π°ΠΊΡ‚Π΅ с Π²Π½ΡƒΡ‚Ρ€ΠΈΠΊΠ»Π΅Ρ‚ΠΎΡ‡Π½Ρ‹ΠΌ растворитСлСм. Π’ настоящСй Ρ€Π°Π±ΠΎΡ‚Π΅ с ΠΏΠΎΠΌΠΎΡ‰ΡŒΡŽ ΠΌΠ΅Ρ‚ΠΎΠ΄Π° ΠœΠΎΠ½Ρ‚Π΅- ΠšΠ°Ρ€Π»ΠΎ Π±Ρ‹Π»ΠΎ ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½ΠΎ ΠΌΠ½ΠΎΠ³ΠΎΠΎΠ±Ρ€Π°Π·ΠΈΠ΅ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½Ρ‹Ρ… ΠΏΠΎΠ»ΠΎΠΆΠ΅Π½ΠΈΠΉ NADPH:FMN-оксидорСдуктазы ΠΎΡ‚Π½ΠΎΡΠΈΡ‚Π΅Π»ΡŒΠ½ΠΎ Π»ΡŽΡ†ΠΈΡ„Π΅Ρ€Π°Π·Ρ‹ с ΡƒΡ‡Π΅Ρ‚ΠΎΠΌ ΠΏΠΎΡ‚Π΅Π½Ρ†ΠΈΠ°Π»Π° элСктростатичСского поля, создаваСмого Π»ΡŽΡ†ΠΈΡ„Π΅Ρ€Π°Π·ΠΎΠΉ. Показано, Ρ‡Ρ‚ΠΎ срСди структур с наимСньшСй энСргиСй взаимодСйствия имССтся комплСкс, Π² ΠΊΠΎΡ‚ΠΎΡ€ΠΎΠΌ ориСнтация Π°ΠΊΡ‚ΠΈΠ²Π½Ρ‹Ρ… Ρ†Π΅Π½Ρ‚Ρ€ΠΎΠ² ΠΎΠ±ΠΎΠΈΡ… Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚ΠΎΠ² способствуСт прямой ΠΏΠ΅Ρ€Π΅Π΄Π°Ρ‡Π΅ Ρ„Π»Π°Π²ΠΈΠ½Π°. Высказано ΠΏΡ€Π΅Π΄ΠΏΠΎΠ»ΠΎΠΆΠ΅Π½ΠΈΠ΅ ΠΎ Ρ€ΠΎΠ»ΠΈ Π²ΠΎΠ΄ΠΎΡ€ΠΎΠ΄Π½ΠΎΠΉ связи ΠΌΠ΅ΠΆΠ΄Ρƒ Arg291 ΠΈ Gln197 Π»ΡŽΡ†ΠΈΡ„Π΅Ρ€Π°Π·Ρ‹ ΠΈ оксидорСдуктазы соотвСтствСнно Π² стабилизации комплСкса ΠΌΠ΅ΠΆΠ΄Ρƒ Π±Π΅Π»ΠΊΠ°ΠΌΠΈA possible mechanism of complex formation between bacterial luciferase and NADPH:FMNoxidoreductase from Vibrio harveyi sustained by electrostatic forces is studied. The complex between the enzymes is important for a direct FMNH2 transfer without a contact with solvent, which could cause a rapid autooxidation and the formation of reactive oxygen species. In the current work the diversity of possible relative positions of NADPH:FMN-oxidoreductase and luciferase was obtained with Monte-Carlo sampling governed by oxidoreductase internal charged groups and electrostatic field caused by luciferase. Among the structures with the minimal energies, the one was found that has a proper active sites orientation for a direct FMNH2 transfer. Possible role of hydrogen bonding between Arg291 and Gln197 of luciferase and oxidoreductase, respectively, in stabilization of this complex is propose

    NAD(P)H:FMN-oxidoreductase functioning under macromolecular crowding: in vitro modeling

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    ВСкст ΡΡ‚Π°Ρ‚ΡŒΠΈ Π½Π΅ публикуСтся Π² ΠΎΡ‚ΠΊΡ€Ρ‹Ρ‚ΠΎΠΌ доступС Π² соотвСтствии с ΠΏΠΎΠ»ΠΈΡ‚ΠΈΠΊΠΎΠΉ ΠΆΡƒΡ€Π½Π°Π»Π°

    Impact of enzyme stabilizers on the characteristics of biomodules for bioluminescent biosensors

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    ВСкст ΡΡ‚Π°Ρ‚ΡŒΠΈ Π½Π΅ публикуСтся Π² ΠΎΡ‚ΠΊΡ€Ρ‹Ρ‚ΠΎΠΌ доступС Π² соотвСтствии с ΠΏΠΎΠ»ΠΈΡ‚ΠΈΠΊΠΎΠΉ ΠΆΡƒΡ€Π½Π°Π»Π°.The biomodule of bioluminescent biosensor based on a coupled enzyme system NADH:FMN-oxidoreductase and luciferase, co-immobilized with substrates in dried starch or gelatin gels, has been developed. We studied the impact of several stabilizers-dithiothreitol (DTT), bovine serum albumin (BSA) and mercaptoethanol (ME) on the biomodule's activity, storage stability and sensitivity to toxic substances. The inclusion of stabilizers increases the activity of the biological module by more than 150%. To achieve the combination of high activity, prolonged storage time and acute sensitivity to toxic substances within maximum permissible concentration we used starch gel as a carrier adding 100 mu M DTT to the immobilized preparation. The gelatin-based biological module had greater storage stability than the starch-based one but demonstrated less sensitivity to toxic substances. (C) 2015 Elsevier B.V. All rights reserved

    Π‘ΠΈΠΎΠ»ΡŽΠΌΠΈΠ½Π΅ΡΡ†Π΅Π½Ρ‚Π½Ρ‹ΠΉ Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π°Ρ‚ΠΈΠ²Π½Ρ‹ΠΉ ΠΈΠ½Π³ΠΈΠ±ΠΈΡ‚ΠΎΡ€Π½Ρ‹ΠΉ Π°Π½Π°Π»ΠΈΠ· наночастиц Π½Π° основС ΠΌΠ΅Ρ‚Π°Π»Π»ΠΎΠ²

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    The bioluminescent enzymatic bioassays for assessment of nanomaterial biotoxicity using the soluble or immobilized coupled enzyme system of luminous bacteria NAD(P)Н:FMN-oxidoreductase + luciferase (Red + Luc) as a test system were employed in this study. This method specifically detects the toxic properties of substances based on their effect on the parameters of the bioluminescent enzyme reactions. The commercially available metal nanoparticles (MNPs), including silver nanoparticles (Ag), nanoparticles of silicon dioxide (SiO2), and titanium dioxide (TiO2), of different sizes were tested in the study. The inhibitory effects of MNPs on the bioluminescent Red + Luc enzyme system were measured. Results indicated that the soluble Red + Luc coupled enzyme system was more sensitive to the inhibition effect of MNPs than its immobilized form. The inhibitory activity of MNPs decreased in the following order: Ag > TiO2 > SiO2. That correlated well with results of other biological methods. Due to substantial advantages such as technical simplicity, short response time and high sensitivity to analysis, this bioluminescent enzymatic bioassay has the potential to be developed as a general bioassay for safety assessment of a wide variety of nanomaterialsΠŸΡ€Π΅Π΄Π»ΠΎΠΆΠ΅Π½ ΠΌΠ΅Ρ‚ΠΎΠ΄ ΠΎΡ†Π΅Π½ΠΊΠΈ биотоксичности Π½Π°Π½ΠΎΠΌΠ°Ρ‚Π΅Ρ€ΠΈΠ°Π»ΠΎΠ², основанный Π½Π° использовании Π² качСствС ΠΎΠ±ΡŠΠ΅ΠΊΡ‚Π° воздСйствия растворимой ΠΈ ΠΈΠΌΠΌΠΎΠ±ΠΈΠ»ΠΈΠ·ΠΎΠ²Π°Π½Π½ΠΎΠΉ Π±ΠΈΠΎΠ»ΡŽΠΌΠΈΠ½Π΅ΡΡ†Π΅Π½Ρ‚Π½ΠΎΠΉ Π±ΠΈΡ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π½ΠΎΠΉ систСмы: НАД(Π€)·Н:ЀМН-оксидорСдуктаза ΠΈ Π»ΡŽΡ†ΠΈΡ„Π΅Ρ€Π°Π·Π°. ΠŸΡ€ΠΈΠ½Ρ†ΠΈΠΏ ΠΌΠ΅Ρ‚ΠΎΠ΄Π° состоит Π² ΠΎΠ±Π½Π°Ρ€ΡƒΠΆΠ΅Π½ΠΈΠΈ токсичСских свойств тСстируСмых вСщСств ΠΏΠΎ ΠΈΡ… влиянию Π½Π° ΠΏΠ°Ρ€Π°ΠΌΠ΅Ρ‚Ρ€Ρ‹ Π±ΠΈΠΎΠ»ΡŽΠΌΠΈΠ½Π΅ΡΡ†Π΅Π½Ρ†ΠΈΠΈ ΠΈΡΠΏΠΎΠ»ΡŒΠ·ΡƒΠ΅ΠΌΠΎΠΉ Π±ΠΈΡ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π½ΠΎΠΉ систСмы. ΠŸΡ€ΠΎΠ²Π΅Π΄Π΅Π½ΠΎ тСстированиС коммСрчСски доступных наночастиц Π½Π° основС ΠΌΠ΅Ρ‚Π°Π»Π»ΠΎΠ² (МНЧ), Π² Ρ‚ΠΎΠΌ числС наночастиц сСрСбра (Ag), ΠΈ Ρ€Π°Π·Π»ΠΈΡ‡Π°ΡŽΡ‰ΠΈΡ…ΡΡ ΠΏΠΎ Ρ€Π°Π·ΠΌΠ΅Ρ€Ρƒ наночастиц диоксидов крСмния (SiO2) ΠΈ Ρ‚ΠΈΡ‚Π°Π½Π° (TiO2). Π­Ρ‚ΠΈ МНЧ ΠΎΠΊΠ°Π·Ρ‹Π²Π°ΡŽΡ‚ ΠΈΠ½Π³ΠΈΠ±ΠΈΡ€ΡƒΡŽΡ‰ΠΈΠΉ эффСкт Π½Π° Π°ΠΊΡ‚ΠΈΠ²Π½ΠΎΡΡ‚ΡŒ Π±ΠΈΡ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π½ΠΎΠΉ систСмы, ΠΏΡ€ΠΈΡ‡Π΅ΠΌ растворимыС Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Ρ‹ Π² большСй стСпСни ΠΏΠΎΠ΄Π²Π΅Ρ€ΠΆΠ΅Π½Ρ‹ ΠΈΠ½Π³ΠΈΠ±ΠΈΡ€ΡƒΡŽΡ‰Π΅ΠΌΡƒ Π²ΠΎΠ·Π΄Π΅ΠΉΡΡ‚Π²ΠΈΡŽ МНЧ ΠΏΠΎ ΡΡ€Π°Π²Π½Π΅Π½ΠΈΡŽ с ΠΈΠΌΠΌΠΎΠ±ΠΈΠ»ΠΈΠ·ΠΎΠ²Π°Π½Π½Ρ‹ΠΌΠΈ. Π‘Ρ‚Π΅ΠΏΠ΅Π½ΡŒ ΠΈΠ½Π³ΠΈΠ±ΠΈΡ€ΡƒΡŽΡ‰Π΅Π³ΠΎ воздСйствия ΡƒΠΌΠ΅Π½ΡŒΡˆΠ°Π΅Ρ‚ΡΡ Π² ряду Ag > TiO2 > SiO2, Ρ‡Ρ‚ΠΎ согласуСтся с Ρ€Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚Π°ΠΌΠΈ Π΄Ρ€ΡƒΠ³ΠΈΡ… биологичСских ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠ². Π‘ΠΈΠΎΠ»ΡŽΠΌΠΈΠ½Π΅ΡΡ†Π΅Π½Ρ‚Π½Ρ‹ΠΉ Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π°Ρ‚ΠΈΠ²Π½Ρ‹ΠΉ ΠΌΠ΅Ρ‚ΠΎΠ΄ Π°Π½Π°Π»ΠΈΠ·Π° Π·Π°Π½ΠΈΠΌΠ°Π΅Ρ‚ 2-3 ΠΌΠΈΠ½, отличаСтся высокой Ρ‡ΡƒΠ²ΡΡ‚Π²ΠΈΡ‚Π΅Π»ΡŒΠ½ΠΎΡΡ‚ΡŒΡŽ, тСхничСской простотой ΠΈ ΠΌΠΎΠΆΠ΅Ρ‚ ΠΈΡΠΏΠΎΠ»ΡŒΠ·ΠΎΠ²Π°Ρ‚ΡŒΡΡ для ΠΎΡ†Π΅Π½ΠΊΠΈ бСзопасности Ρ€Π°Π·Π»ΠΈΡ‡Π½Ρ‹Ρ… классов Π½Π°Π½ΠΎΠΌΠ°Ρ‚Π΅Ρ€ΠΈΠ°Π»ΠΎ
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