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

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

Bioluminescent System of Luminous Bacteria for Detection of Microbial Contamination

Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5’-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FM

Bioluminescent enzyme inhibition based assay of metal nanoparticles

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Applications of luminous bacteria enzymes in toxicology

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

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

Биолюминесцентный ферментативный ингибиторный анализ наночастиц на основе металлов

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 nanomaterialsПредложен метод оценки биотоксичности наноматериалов, основанный на использовании в качестве объекта воздействия растворимой и иммобилизованной биолюминесцентной биферментной системы: НАД(Ф)·Н:ФМН-оксидоредуктаза и люцифераза. Принцип метода состоит в обнаружении токсических свойств тестируемых веществ по их влиянию на параметры биолюминесценции используемой биферментной системы. Проведено тестирование коммерчески доступных наночастиц на основе металлов (МНЧ), в том числе наночастиц серебра (Ag), и различающихся по размеру наночастиц диоксидов кремния (SiO2) и титана (TiO2). Эти МНЧ оказывают ингибирующий эффект на активность биферментной системы, причем растворимые ферменты в большей степени подвержены ингибирующему воздействию МНЧ по сравнению с иммобилизованными. Степень ингибирующего воздействия уменьшается в ряду Ag > TiO2 > SiO2, что согласуется с результатами других биологических методов. Биолюминесцентный ферментативный метод анализа занимает 2-3 мин, отличается высокой чувствительностью, технической простотой и может использоваться для оценки безопасности различных классов наноматериало

Applications of luminous bacteria enzymes in toxicology

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