Motivation: Bacterial luciferases are heterodimeric enzymes that catalyze a chemical reaction, so called bioluminescence, which causes light emission in bacteria. Bioluminescence is vastly used as a reporter system in research tools and commercial developments. However, the details of the mechanisms that stabilize and transform the reaction intermediates as well as differences in the enzymatic kinetics amongst different bacterial luciferases remain to be elucidated.

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 $\alpha$Glu175 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.

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.

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.