The crucial role of Y109 and R162 as catalytic residues of nanoKAZ: insights from molecular docking, molecular dynamics simulation, and quantum chemical investigations.

CONTEXT: nanoKAZ is a compact luciferase that exhibits intense blue light emission when it catalyzes the substrate Furimazine (FMZ) as a luciferin, making it an excellent candidate as a reporter protein. However, the specific catalytic residues and mechanism of nanoKAZ have not been revealed. Recently, the structure of nanoKAZ was determined, and it was observed that the luminescent properties changed when FMZ analogs with naphthalene replacing benzene were used. It is speculated that the substituted naphthalene may influence the interaction between the catalytic residues and luciferins, thereby affecting the energy of the emitted light signal. METHOD: Therefore, the primary objective of this study is to analyze and compare the molecular recognition between nanoKAZ and FMZ along with its four activity-altered naphthalene analogs, with aiming to identify the catalytic residues. Molecular docking was employed to construct all nanoKAZ-luciferin models, followed by a 500 ns molecular dynamics simulation. The simulation trajectory was subjected to MM/PBSA analysis to identify crucial residues that contribute significantly to luciferin binding. In the result, two polar residues Y109, and R162 were identified as active residues as their notable contributions to the binding energy. Subsequently, an oxygen molecule was introduced into the local region of the nanoKAZ-FMZ complex and followed with quantum chemical calculations (semiempirical and DFT methods were used) to investigate the catalysis details. The results illustrated the involvement of Y109 and R162 in the oxygenation of FMZ, leading to the formation of dioxetanone, which has been suggested as an important intermediate in the oxidation process among various luciferins sharing the same functional group as FMZ.