Viscous Media Slow Down the Decay of the Key Intermediate in Bacterial Bioluminescent Reaction

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

doi:10.1134/s1607672920020106

Cited by 6 publications

(21 citation statements)

References 14 publications

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“…In increasing viscosity of the medium, both substrate-binding steps are slowed down in a similar way and demonstrated the enhanced effect implying contribution of additional factors, not only diffusion control. Taking into account that viscosity effect on such competitive reaction steps as flavin autooxidation and peroxyflavin intermediate dark decay did not exhibit any enhancement, as it was shown earlier in [28], we can conclude that higher viscosity provides no kinetic advantages for the "light" reaction pathways.…”

Section: Discussionsupporting

confidence: 63%

“…With a minimization parameter based on the least-squares fit error, the program searched for the best values of intensity at each time step that most accurately matched the experimental data (Figure S1C,D). Two decay constants (k d , k dd ) were fixed during simulation because they were determined in particular experiments [28]. Bioluminescence kinetic curves obtained at five different concentrations of decanal (10, 20, 30, 40 and 50 µM) in the buffer and in the presence of sucrose or glycerol were used as input data and simultaneously simulated.…”

Section: Mathematical Modelling Of the Reaction Kineticsmentioning

confidence: 99%

“…Two of them (k d , k dd ) were preliminarily estimated for each viscous solution in the particular experiments as it is described in [28]. Therefore, they were fixed during the modeling sessions.…”

Section: Mathematical Modeling Of the Kinetics Of The Bacterial Bioluminescent Reactionmentioning

confidence: 99%

“…1. # The values of k d and k dd have been taken from [28] and fixed during the fitting session. The values for 30% glycerol have been obtained by interpolation.…”

Section: Mathematical Modeling Of the Kinetics Of The Bacterial Bioluminescent Reactionmentioning

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: Discussionsupporting

confidence: 63%

Section: Mathematical Modelling Of the Reaction Kineticsmentioning

confidence: 99%

Section: Mathematical Modeling Of the Kinetics Of The Bacterial Bioluminescent Reactionmentioning

confidence: 99%

“…1. # The values of k d and k dd have been taken from [28] and fixed during the fitting session. The values for 30% glycerol have been obtained by interpolation.…”

Section: Mathematical Modeling Of the Kinetics Of The Bacterial Bioluminescent Reactionmentioning

confidence: 99%

See 2 more Smart Citations

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

Lisitsa

Sukovatyi

Барцев

et al. 2021

IJMS

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“…The contribution of k dd into k decay substantially differs for two luciferases. In reaction with decanal at 20 °C, the k decay values are 0.32 and 0.21 s −1 for the V. harveyi and P. leiognathi enzymes, whereas the corresponding k dd are 0.06 and 0.15 s −1 [ 33 , 34 ]. Thus, distinct temperature dependences of k decay for two luciferases could be caused by two reasons: (i) by a stronger destabilizing effect on peroxyflavin intermediate in V. harveyi reaction and/or (ii) by temperature influence on the others than k dd rate constants which could be more pronounced in the V. harveyi reaction due to their high contribution into k decay .…”

Section: Discussionmentioning

confidence: 99%

Structure-Function Relationships in Temperature Effects on Bacterial Luciferases: Nothing Is Perfect

Deeva

Lisitsa

Sukovatyi

et al. 2022

IJMS

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The evaluation of temperature effects on the structure and function of enzymes is necessary to understand the mechanisms underlying their adaptation to a constantly changing environment. In the current study, we investigated the influence of temperature variation on the activity, structural dynamics, thermal inactivation and denaturation of Photobacterium leiognathi and Vibrio harveyi luciferases belonging to different subfamilies, as well as the role of sucrose in maintaining the enzymes functioning and stability. We used the stopped-flow technique, differential scanning calorimetry and molecular dynamics to study the activity, inactivation rate, denaturation and structural features of the enzymes under various temperatures. It was found that P. leiognathi luciferase resembles the properties of cold-adapted enzymes with high activity in a narrow temperature range and slightly lower thermal stability than V. harveyi luciferase, which is less active, but more thermostable. Differences in activity at the studied temperatures can be associated with the peculiarities of the mobile loop conformational changes. The presence of sucrose does not provide an advantage in activity but increases the stability of the enzymes. Differential scanning calorimetry experiments showed that luciferases probably follow different denaturation schemes.

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Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

Pimviriyakul

Chaiyen

2022

The FEBS Journal

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HadA monooxygenase catalyses the detoxification of halogenated phenols and nitrophenols via dehalogenation and denitration respectively. C4a‐hydroperoxy‐FAD is a key reactive intermediate wherein its formation, protonation and stabilization reflect enzyme efficiency. Herein, transient kinetics, site‐directed mutagenesis and pH‐dependent behaviours of HadA reaction were employed to identify key features stabilizing C4a‐adducts in HadA. The formation of C4a‐hydroperoxy‐FAD is pH independent, whereas its decay and protonation of distal oxygen are associated with pKa values of 8.5 and 8.4 respectively. These values are correlated with product formation within a pH range of 7.6–9.1, indicating the importance of adduct stabilization to enzymatic efficiency. We identified Arg101 as a key residue for reduced FAD (FADH−) binding and C4a‐hydroperoxy‐FAD formation due to the loss of these abilities as well as enzyme activity in HadAR101A and HadAR101Q. Mutations of the neighbouring Asn447 do not affect the rate of C4a‐hydroperoxy‐FAD formation; however, they impair FADH− binding. The disruption of Arg101/Asn447 hydrogen bond networking in HadAN447A increases the pKa value of C4a‐hydroperoxy‐FAD decay to 9.5; however, this pKa was not altered in HadAN447D (pKa of 8.5). Thus, Arg101/Asn447 pair should provide important interactions for FADH− binding and maintain the pKa associated with H2O2 elimination from C4a‐hydroperoxy‐FAD in HadA. In the presence of substrate, the formation of C4a‐hydroxy‐FAD at the hydroxylation step is pH insensitive, and it dehydrates to form the oxidized FAD with pKa of 7.9. This structural feature might help elucidate how the reactive intermediate was stabilized in other flavin‐dependent monooxygenases.

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Viscous Media Slow Down the Decay of the Key Intermediate in Bacterial Bioluminescent Reaction

Cited by 6 publications

References 14 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

Structure-Function Relationships in Temperature Effects on Bacterial Luciferases: Nothing Is Perfect

Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

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