TY - JOUR

T1 - A suicidal and extensively disordered luciferase with a bright luminescence

AU - Dijkema, Fenne Marjolein

AU - Escarpizo-Lorenzana, Marta Iglesia

AU - Nordentoft, Matilde Knapkøien

AU - Rabe, Hanna Christin

AU - Sahin, Cagla

AU - Landreh, Michael

AU - Branca, Rui Mamede

AU - Sørensen, Esben Skipper

AU - Christensen, Brian

AU - Prestel, Andreas

AU - Teilum, Kaare

AU - Winther, Jakob Rahr

N1 - Publisher Copyright: © 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.

PY - 2024

Y1 - 2024

N2 - Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

AB - Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

KW - active site

KW - enzyme

KW - intrinsic disorder

KW - luciferase

KW - NMR

KW - oxidation

KW - structure

U2 - 10.1002/pro.5115

DO - 10.1002/pro.5115

M3 - Journal article

AN - SCOPUS:85198725705

VL - 33

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 8

M1 - e5115

ER -