The Effect of Osmolytes on the Bioluminescent Reaction of Bacteria: Structural and Dynamic Properties

Sukovatyi, Lev A.; Lisitsa, Albert E.; Kratasyuk, Valentina A.; Nemtseva, Elena

doi:10.1134/s0006350920060202

Cited by 4 publications

(13 citation statements)

References 19 publications

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“…Additionally, it was found that the functionally important mobile loop of luciferase (262-291 a. r.) demonstrated higher mobility at low concentration of the cosolvents (≤10%). Also previously we estimated the amount of water and cosolvent molecules in the active site of bacterial luciferase as an average value by the last 20 ns of the molecular dynamics modeling [30] and revealed that it can contain glycerol molecules but not sucrose. In the current study, we analyzed conformational preferences of amino acid residues responsible for flavin and aldehyde binding, their involvement in H-bonding with cosolvent molecules and penetration of cosolvents into the active site gorge.…”

Section: Discussionmentioning

confidence: 96%

“…To elucidate the mechanisms of the found specific effects we used classical all-atom molecular dynamics simulation in explicit solvent, which is routinely applied to reveal the peculiarities of protein-cosolvent interaction [11]. Earlier we showed that no significant conformational changes of bacterial luciferase occur in solutions with 10-40% of sucrose or glycerol, which was confirmed by unchanged RMSD (root mean square deviation) and SASA (solvent accessible surface area) of the protein [30]. Additionally, it was found that the functionally important mobile loop of luciferase (262-291 a. r.) demonstrated higher mobility at low concentration of the cosolvents (≤10%).…”

Section: Discussionmentioning

confidence: 99%

“…Before starting the reaction, FMN was photoreduced by exposure of the Solution A under the light of an incandescent lamp for 10 min. For experiments in viscous media, the mixtures of the buffer with glycerol or sucrose (10,20,30,40 (w/w)%) were used as a solvent. The kinetics of bioluminescence was recorded under temperature control (at 20 • C) for 15 s with a photomultiplier directly placed on the observation cell of the spectrometer without additional filters.…”

Section: Methodsmentioning

confidence: 99%

“…The structure was surrounded by water molecules and mixtures with glycerol or sucrose molecules adjusted to simulate the media with 10, 20, 30, 40 (w/w)% of cosolvent (Table S1). The details of MD simulation can be found in [30] and in the Supplementary Material. CASTp web-service was used to determine flavin and aldehyde binding cavity within the crystal structure of V. harveyi luciferase [31].…”

Section: Computational Analysis Of Bacterial Luciferase-cosolvents Interactionsmentioning

confidence: 99%

“…The molecular dynamics of V. harveyi luciferase surrounded by water molecules or mixtures of water with sucrose/glycerol was simulated in three independent runs of 40 ns each [30]. In the current study, we analyzed local structural and dynamic properties of the protein that alter in the presence of the cosolvents.…”

Section: Effects Of Glycerol and Sucrose Molecules On Bacterial Luciferase Structurementioning

confidence: 99%

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Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Lisitsa

Sukovatyi

Барцев

et al. 2021

IJMS

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Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial bioluminescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. Moreover, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Computational Analysis Of Bacterial Luciferase-cosolvents Interactionsmentioning

confidence: 99%

Section: Effects Of Glycerol and Sucrose Molecules On Bacterial Luciferase Structurementioning

confidence: 99%

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Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Lisitsa

Sukovatyi

Барцев

et al. 2021

IJMS

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Coupling of NAD(P)H:FMN‐oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain

Sutormin,

Nemtseva,

Gulnov

et al. 2023

Photochem & Photobiology

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The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN‐oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5–20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc‐Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.

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Effect of Viscous Media on the Photophysical Characteristics of Flavin Mononucleotide

Gulnov

Gerasimova

Sukovatyi

et al. 2022

Bull. Russ. Acad. Sci. Phys.

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The Effect of Osmolytes on the Bioluminescent Reaction of Bacteria: Structural and Dynamic Properties

Cited by 4 publications

References 19 publications

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Coupling of NAD(P)H:FMN‐oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain

Effect of Viscous Media on the Photophysical Characteristics of Flavin Mononucleotide

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