Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelicafirefly luciferase

Abstract

Insufficient thermal stability of firefly luciferases often limits their application in a wide range of fields. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases. However, for Hotaria parvulafirefly luciferase, that shares 98% degree of homology with Luciola mingrelicaluciferase, the A217L mutation is known to dramatically decrease catalytic activity. We analyzed the environment of A217 in the 3D-structure of L. mingrelicaluciferase with the purpose of identifying possible additional mutations that would allow retention of the high thermal stability of the A217L mutant while preserving the high level of activity. The G216N/A217L double mutant of L. mingrelicaluciferase demonstrated significantly improved stability at 42 and 45 °C but retained only 10% of activity; the loss in activity was accompanied by a large red shift of bioluminescence emission maximum from 566 to 611 nm compared with the wild-type enzyme. The triple mutant G216N/A217L/S398M exhibited high thermal stability of the double mutant together with the high activity and bioluminescence spectra close to that of the wild-type luciferase.