title: Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelica firefly 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 parvula firefly luciferase, that shares 98% degree of homology with Luciola mingrelica luciferase, the A217L mutation is known to dramatically decrease catalytic activity. We analyzed the environment of A217 in the 3D-structure of L. mingrelica luciferase 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. mingrelica luciferase 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.
authors.affiliation: Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia. mkoksharov@gmail.com
authors.forename: Mikhail I
authors.initials: MI
authors.lastname: Koksharov
authors.forename: Natalia N
authors.initials: NN
authors.lastname: Ugarova
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D048888:Fireflies / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D049409:Luciferases, Firefly