Bacterial luciferase is a member of the large family of flavin monooxygenases, among which it performs the unique bioluminescent reaction. We studied the interaction of two bacterial luciferases (Vibrio harveyi - of "slow" type and Photobacterium leiognathi - of "fast" type) with aliphatic aldehydes using experimental kinetic techniques, molecular docking, molecular dynamics and phylogenetic analysis. The aim was to understand the structural basis for the different kinetic properties of the two types of luciferase with aldehyde substrate. We found that a distinctive feature of the active site of V. harveyi luciferase was the presence of a minor aldehyde-binding pocket. In P. leiognathi luciferase, all aldehydes bound to the hydrophobic site of the active center in approximately the same conformation, differing only in the binding affinity. In V. harveyi luciferase, aldehydes C8 and C12 did not interact with αTrp194, consistent with their slow reaction kinetics. The molecular dynamics and free energy calculations indicated that the hydrophobic interaction between the aldehyde and protein is the driving force for the complex stability. Phylogenetic analysis of bacterial luciferase sequences revealed that amino acids from the aldehyde binding site at positions 6-8, 191-195, and 228-229 are under positive evolutionary selection. These data indicate that evolutionary processes have shaped a universal platform for binding the natural aldehyde tetradecanal, enabling efficient catalysis in both "fast" and "slow" luciferases. However, when shorter aldehydes are used in bioluminescent assays, the specific features of their interactions within the active site must be taken into account, as they determine the kinetics of the enzyme.

7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF or F0) is a chromophore involved into electronic energy transfer processes in DNA-photolyases, cryptochromes and bioluminescent reaction of the Siberian earthworm, Henlea sp. We studied the impact of the solvent properties (polarity, hydrogen bonding ability, etc.) on F0 fluorescence using the Catalan multiparametric approach and molecular dynamics technique. Analysis of the absorption and fluorescence spectra, as well as fluorescence decays and decay-associated spectra, of F0 in 14 solvents revealed the presence of a mixture of protonated and deprotonated forms - F0H and F0-, respectively, in the majority of solvents. We performed a correlation analysis of the spectral maxima of excitation/emission spectra and fluorescence lifetimes with the Catalan parameters of the solvents. A stronger dependence of spectral maxima of F0- than of F0H on the solvent polarizability (SP) was found. For both F0H and F0-, a weaker effect of solvent basicity (SB) was oppositely directed on excitation and emission spectra, which resulted in a stronger effect on the Stokes shifts. In water solutions with low pH (2.9-4.5), absorption and fluorescence spectra of F0 indicated the proton transfer during the excited-state lifetime (ESPT). For this process, ΔpKa = 3.67 ± 0.04 was first obtained, characterizing F0 as a weak photoacid. All-atom molecular dynamics simulations of F0H and F0- in water, methanol, DMSO, and acetone demonstrated that solvent hydrogen-bonding interactions influence the fluorophore's conformational dynamics and could alter F0 photophysical properties. A special role of the N3 atom as a donor of the hydrogen bonds with solvents or proteins was proposed.

The structural dynamics of two proteins—luciferases from bacteria Vibrio harveyi and Photobacterium leiognathi, was studied using time-resolved fluorescence and phosphorescence of the tryptophan residues at 5–35°C. Earlier, the temperature effects on functional properties of these luciferases were found to be different. Revealing the structural and dynamic basis of the difference will contribute to understanding the principles of molecular adaptation of cells for living in unfavorable conditions. Nanosecond-resolved shifts of the fluorescence spectra were analyzed and dipolar relaxation times of the tryptophans microenvironment were obtained: τR1 = 0.22–0.49 ns and τR2 = 8.04–9.37 ns for P. leiognathi luciferase and τR1 = 0.18–0.27 ns and τR2 = 6.3–9.0 ns for V. harveyi luciferase. Temperature dependences of τR1 indicate a more rigid structure of the latter protein. Microsecond-resolved phosphorescence decays were measured and phosphorescence lifetimes τp1 and τp2 were determined: 0.15–0.30 and 0.91–2.77 ms for P. leiognathi luciferase and 0.16–0.33 and 2.27–5.45 ms for V. harveyi luciferase, respectively. The increased values of τp2 for V. harveyi luciferase also indicate more rigid microenvironment of the tryptophans within this protein. The experimental data were compared with the molecular modeling results on the mobility of tryptophans and adjacent residues in bacterial luciferase structures under temperature variations.

Based on the data on the transient kinetics of the bioluminescent reaction catalyzed by P. leiognathi luciferase in media with polyols and sugars, the relative quantum yield of bioluminescence in this reaction per substrate molecule was determined using mathematical modeling. It was obtained that in some media the relative quantum yield per aldehyde molecule increases compared to the value in the buffer: by 18 and 33

In this work, we considered the influence of viscogenic agents (glycerol, sucrose) as well as the temperature on the fluorescent characteristics of fluorescein at pH 6.5 in order to describe the acid-base status of local environment in terms of a spectrally detectable dianion-anion equilibrium. The protolytic equilibrium of fluorescein was found to depend on the solvent viscosity in a complex way. Whereas in the presence of sucrose the ratiometric signal of fluorescein (I488/I435) remains rather unchanged, the addition of glycerol (up to 40% w/w) results in the increase of the signal (up to 19%), that can be attributed to the different mechanisms of cosolvents effects on dye molecules in the ground state. Molecular dynamics of the dye in the presence of glycerol and sucrose revealed that the cosolvents preferentially interact with fluorescein monoanion and dianion, displacing water molecules from the local environment which in turn reduces the average number of the hydrogen bonds between xanthene ring of the dye and water molecules. The ratiometric signal demonstrates linear growth with the temperature in the range of 10–80 °C regardless of the presence of viscogenic agents. A linear correlation between the temperature sensitivity of the ratiometric signal and the change in the molar enthalpy of the proton dissociation reaction in buffer and viscous media was determined.

The present work is a review of the research on using hydrogels based on natural biodegradable polymers, starch, and gelatin for enzyme immobilization. This review addresses the main properties of starch and gelatin that make them promising materials in biotechnology for producing enzyme preparations stable during use and storage and insensitive to chemical and physical impacts. The authors summarize their achievements in developing the preparations of enzymes immobilized in starch and gelatin gels and assess their activity, stability, and sensitivity for use as biorecognition elements of enzyme inhibition-based biosensors.

Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that "inert" ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms.

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.

This study examined the effects of glycerol and sucrose on the coupled reactions of NAD(P)H:FMN-oxidoreductase of luminous bacteria in vitro, providing valuable information regarding microenvironment of the enzyme and its function.

Chemoenzymatic assay systems are widely used to detect toxicants in various samples, including food and environment specimens. These methods are based on the ability of various types of toxicant to specifically inhibit/activate the functions of individual enzymes or enzyme systems. The present study examines the possibility of using the proteolytic enzyme trypsin as a specific marker to detect protease inhibitors in different samples. The study shows that trypsin activity is not affected by various heavy metals, pesticides, or quinones at levels considerably greater than their maximum allowable concentrations (MACs) in water bodies. At the same time, the IC50 value for the food preservative potassium sorbate (E202) is 15 mg/L, which is substantially lower than its acceptable daily intake (ADI). The quenching of trypsin fluorescence in the presence of potassium sorbate suggests that inhibition could occur due to the binding of the preservative to the enzyme in the region adjacent to the active center. The trypsin was immobilized in starch gel to ensure its stability in the enzyme inhibition based assay. Single-use reagents were prepared as dry starch disks that could be stored over long periods. Their sensitivity to copper (II) chloride, potassium sorbate, and chromium (III) chloride was similar to the sensitivity of the free trypsin.

A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water-cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site.

Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.

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.

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

A study is performed of the absorption and luminescence of flavin mononucleotide in viscous media with different contents of glycerol or sucrose. It is shown that in addition to the general effects of polarity and viscosity, the spectral characteristics of flavin are determined by specific interactions between sucrose and the isoalloxazine ring.

Nowadays the recombinant Ca2+-regulated photoproteins originating from marine luminous organisms are widely applied to monitor calcium transients in living cells due to their ability to emit light on Ca2+ binding. Here we report the specific activities of the recombinant Ca2+-regulated photoproteins-aequorin from Aequorea victoria, obelins from Obelia longissima and Obelia geniculata, clytin from Clytia gregaria and mitrocomin from Mitrocoma cellularia. We demonstrate that along with bioluminescence spectra, kinetics of light signals and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of bioluminescent reactions. The highest specific activities were found for obelins and mitrocomin, whereas those of aequorin and clytin were shown to be lower. To determine the factors influencing the variations in specific activities the fluorescence quantum yields for Ca2+-discharged photoproteins were measured and found to be quite different varying in the range of 0.16-0.36. We propose that distinctions in specific activities may result from different efficiencies of singlet excited state generation and different fluorescence quantum yields of coelenteramide bound within substrate-binding cavity. This in turn may be conditioned by variations in the amino acid environment of the substrate-binding cavities and hydrogen bond distances between key residues and atoms of 2-hydroperoxycoelenterazine.

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.

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.

Coelenterazine-v (CTZ-v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca2+ -regulated photoproteins activated with CTZ-v also display red-shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ-v unusable for calcium imaging. Here, we report the crystal structure of Ca2+ -regulated photoprotein obelin with 2-hydroperoxycoelenterazine-v (obelin-v) at 1.80 Å resolution. The structures of obelin-v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen-bond pattern and slightly increased distances between key active site residues and some atoms of 2-hydroperoxycoelenterazine-v were found. The fluorescence quantum yield (ΦFL ) of obelin bound with coelenteramide-v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme-substrate complexes containing the 2-hydroperoxy adduct of CTZ-v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (ΦR ) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin-v is caused by the low efficiency of generating an electronic excited state (ΦS ). In turn, the low ΦS value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin-v active site.

Исследована скорость распада интермедиата биолюминесцентной реакции бактерий 4a-гидропероксифлавина в зависимости от вязкости среды.Обнаружено, что при низких концентрациях глицерина и сахарозы (вязкость 1.1-1.3сП) скорость распада увеличивается, а дальнейший рост вязкости до 6.2 сП приводит к уменьшению скорости распада по степенному закону с показателем 0.82-0.84.Методами молекулярной динамики установлено, что в средах с глицерином и сахарозой происходит изменение подвижности аминокислотных остатков активного центра люциферазы, отвечающих за связывание флавина.Полученные результаты говорят о двух разнонаправленных эффектах вязких сред с добавлением глицерина и сахарозы: 1) дестабилизация 4a-гидропероксифлавина за счет изменения структурно-динамических свойств белка, 2) стабилизация этого интермедиата за счет снижения скорости диффузии продуктов его распада.