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

Abstract

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.

The Role of Cosolvent–Water Interactions in Effects of the Media on Functionality of Enzymes: A Case Study of Photobacterium leiognathi Luciferase

“…Enzymes from two subfamilies (such as Vibrio harveyi and Photobacterium leiognathi ) differ slightly in the architecture of the active site gorge and in the mode of substrate binding, as well as in the composition of the functional mobile loop. Our previous research showed that they are also characterized by distinct dynamic properties [ 26 , 27 ]. Therefore, in the present study, the homology-modeled structure of P. leiognathi luciferase was used instead of the resolved crystal structure of V. harveyi luciferase.…”

“…The all-atom molecular dynamics (MD) of bacterial luciferase surrounded by water and cosolvents was simulated using GROMACS 2020.04 with CHARMM36 force field [ 50 , 51 ]. In order to model specific interactions of P. leiognathi luciferase surface and functional loops with cosolvents, its initial coordinates were obtained via homology-based modeling as described in [ 27 ]. Luciferase was modeled without any of the reaction components and their derivatives, since their structures in complex with the enzyme have not been crystalized yet (except for the product of the reaction [ 52 ]), and conformational changes in the protein were the focus of this study.…”

“…We conducted three independent 100 ns molecular dynamics simulations for each system; the parameters and modeling routine can be found elsewhere [ 27 ]. In all simulations, the temperature was kept constant at T = 300 K. The numbers of cosolvent/water molecules in model boxes with cosolvents were: ethylene glycol (30 wt %)—3694/28,546, glycerol (40 wt %)—3320/25,958, sorbitol (40 wt %)—1678/27,419, glucose (40 wt %)—1697/28,241, and sucrose (40 wt %)—893/28,968.…”