Structural and evolutionary analysis of aldehyde binding sites in two types of bacterial luciferase in relation to reaction kinetics.

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.